METHOD OF PRODUCING RECOMBINANT HIGH MOLECULAR WEIGHT vWF IN CELL CULTURE

ABSTRACT

Among other aspects, the present invention relates to cell culture conditions for producing high molecular weight vWF, in particular, highly multimericWF with a high specific activity and ADAMTS13 with a high specific activity. The cell culture conditions of the present invention can include, for example, a cell culture medium with an increased copper concentration and/or cell culture supernatant with a low ammonium (NH 4   + ) concentration. The present invention also provides methods for cultivating cells in the cell culture conditions to express high molecular weight vWF and rA13 having high specific activities.

CROSS REFERENCES TO APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.16/125,141, filed Sep. 7, 2018, (now issued as U.S. Pat. No.10,822,394), which is a Continuation of U.S. patent application Ser. No.15/591,022, filed May 9, 2017, (now issued as U.S. Pat. No. 10,100,099),which is a Continuation of U.S. patent application Ser. No. 15/201,125,filed Jul. 1, 2016, (now issued as U.S. Pat. No. 9,834,591), which is aDivisional of U.S. patent application Ser. No. 14/339,319, filed Jul.23, 2014, (now issued as U.S. Pat. No. 9,409,971), which is a Divisionalof U.S. patent application Ser. No. 13/179,386, filed Jul. 8, 2011 (nowissued as U.S. Pat. No. 8,852,888), which claims priority to U.S.Provisional Patent Application Ser. No. 61/362,635, filed Jul. 8, 2010,the disclosures of which are hereby incorporated herein by reference intheir entireties for all purposes.

BACKGROUND OF THE INVENTION

The recombinant expression of therapeutic proteins in cell culture(particularly large-scale cell cultures), including eukaryotic cellculture, and more specifically mammalian cell culture, requires the useof special culture media providing nutrient substances for efficientgrowth of cells. Cell culture media formulations are often supplementedwith a range of additives, including fetal calf serum (FCS), animalderived proteins and/or protein hydrolysates of bovine origin as well asprotein hydrolysates derived from plants or yeast. One challenge withsuch cultures is that the amount of protein and the total and specificactivity of the protein produced are often variable across differentcell cultures, even when the formulation for the cell culture media isnot changed. This variability is especially apparent in the case oflarge-scale manufacturing processes utilizing cell culture volumes of 10liters to over 20,000 liters. Cell culture media containing hydrolysatesare particularly prone to variability from one cell culture to the next,leading to decreased production of total protein as well as decreasedtotal and specific activity.

One potential reason for the variability seen across different cellcultures is that contaminants in additives such as hydrolysates varyfrom one batch to the next. In general, serum or serum-derivedsubstances, such as, e.g., albumin, transferrin or insulin, may compriseunwanted agents that can contaminate the cell cultures and thebiological products obtained thereof. Furthermore, human serum derivedadditives have to be tested for all known viruses, including hepatitisviruses and HIV which can be transmitted via serum. Moreover, bovineserum and products derived thereof bear the risk of BSE contamination.In addition, all serum- derived products can be contaminated by unknownsubstances. When using serum or protein additives derived from human oranimal sources in cell culture, there are numerous problems (e.g., thevarying quality in composition of different batches and the risk ofcontamination with mycoplasma, viruses or BSE), particularly if thecells are used in the manufacture of drugs or vaccines for humanadministration. Therefore, many attempts have been made to provideefficient host systems and cultivation conditions, which do not requireserum or other animal protein compounds.

Such serum-free media have been developed on the basis of proteinextracts derived from plants or yeast. For example, soy hydrolysates areknown to be useful for fermentation processes and can enhance the growthof many fastidious organisms, yeasts and fungi. WO 96/26266 describesthat papaic digests of soy meal are a source of carbohydrate andnitrogen and many of the components can be used in tissue culture.Franek et al. (Biotechnology Progress (2000) 16, 688-692) describegrowth and productivity promoting effects of defined soy and wheathydrolysate peptide fractions.

WO 96/15231 discloses a serum-free medium composed of a syntheticminimal essential medium and a yeast extract for the propagation ofvertebrate cells and a virus production process. A medium formulationcomposed of a basal cell culture medium comprising a rice peptide and anextract of yeast and an enzymatic digest thereof, and/or a plant lipidfor growth of animal cells is disclosed in WO 98/15614. A mediumcomprising purified soy hydrolysate for the cultivation of recombinantcells is disclosed in WO 01/23527. WO 00/03000 discloses a medium thatcomprises a soy hydrolysate and a yeast extract, but also requires thepresence of recombinant forms of animal proteins, such as growthfactors.

EP-A-0 481 791 describes a biochemically defined culture medium forculturing engineered CHO cells, which is free from protein, lipid andcarbohydrate isolated from an animal source, further comprising arecombinant insulin or insulin analogue, 1% to 0.025% w/v papaindigested soy peptone and putrescine. WO 98/08934 describes a serum-freeeukaryotic cell culture comprising hydrolyzed soy peptides (1-1000mg/L), 0.01 to 1 mg/L putrescine and a variety of animal-derivedcomponents, including albumin, fetuin, various hormones and otherproteins. In this context, it should be noted that putrescine is alsoknown to be comprised in standard media like DMEM/Ham's F12 in aconcentration of 0.08 mg/L.

The plant and/or yeast hydrolysates, however, are undefined mixtures ofoligopeptides and other unknown components and contaminants. Moreover,the quality of commercially available lots of hydrolysates variesextremely. As a result, there are large variations in the production ofrecombinant proteins or viral products (a variation of up to a factor ofthree) as a function of the lots of hydrolysates used (“lot-to-lotvariation”). This drawback affects the proliferation of the cells aswell as the protein expression of each cell. US 2007/0212770 describesvarious animal protein-free and oligopeptide-free, chemically definedculture mediums that are useful for the large-scale production ofrecombinant protein biopharmaceuticals.

Hemostasis involves the interaction of various hemostatic reactionroutes finally leading to thrombus formation. Thrombi are deposits ofblood components on the surface of the vascular wall that mainly consistof aggregated blood platelets and insoluble cross-linked fibrin. Fibrinformation is the result of the restricted proteolysis of fibrinogen bythrombin, a coagulation enzyme. Thrombin is the end product of thecoagulation cascade, a succession of zymogen activations occurring onthe surfaces of activated blood platelets and leucocytes, and a varietyof vascular cells (for a survey, cf. K. G. Mann et al., Blood, 1990,Vol. 76, pp. 1-16).

An important function in the coagulation cascade resides in theactivation of Factor X by the complex of activated Factor IX (FactorIXa) and activated Factor VIII (Factor VIIIa). A deficiency or adysfunction of the components of this complex is associated with theblood disease known as hemophilia (J. E. Sadler & E. W. Davie:Hemophilia A, Hemophilia B, and von Willebrand's Disease, in G.Stamatoyannopoulos et al., (Eds.): The molecular basis of blooddiseases. W.B. Saunders Co., Philadelphia, 1987, pp. 576-602).Hemophilia A is related to a deficiency of Factor VIII activity, whereasHemophilia B is related to a Factor IX deficiency. Current treatmentconsists of a replacement therapy using pharmaceutical preparationscomprised of the normal coagulation factor. Of these thrombopathies,Hemophilia A occurs more frequently, affecting approximately one out of10,000 men. Replacement therapy in Hemophilia A patients involves therepeated administration of preparations containing normal Factor VIII byintravenous infusion. The interval between the infusions is a functionof the degradation of the Factor VIII activity in blood circulation. Thehalf-life of the Factor VIII activity after an infusion differs from oneindividual to another, ranging from 10 to 30 hours. Thus, a prophylactictherapy requires an infusion every two to three days. This constitutes aheavy load on the life of hemophilic patients, in particular, if thevenous access has become difficult due to local citratization followingfrequent needle punctures for intravenous infusions.

It would be particularly advantageous if the frequency of infusionscould be lowered by using Factor VIII having extended half-lives. It iswell known in the art that the half-life of the non-activated FactorVIII heterodimer strongly depends on the presence of von WillebrandFactor, which exhibits a strong affinity to Factor VIII (yet not toFactor VIIIa) and serves as a carrier protein (J. E. Sadler and E. W.Davie: Hemophilia A, Hemophilia B and von Willebrand's disease, in G.Stamatoynnopoulos et al. (Eds.): The molecular basis of blood diseases.W.B. Saunders Co., Philadelphia, 1987, pp. 576-602). It is known thatpatients suffering from von Willebrand's disease type 3, who do not havea detectable von Willebrand Factor in their blood circulation, alsosuffer from a secondary Factor VIII deficiency. In addition, thehalf-life of intravenously administered Factor VIII in those patients is2 to 4 hours, which is considerably shorter than the 10 to 30 hoursobserved in Hemophilia A patients. From these findings results thatFactor VIII tends to a rapid clearance from the blood circulation andthat this process is to some extent inhibited by complexation with itsnatural carrier, von Willebrand Factor.

Von Willebrand factor (vWF) is a glycoprotein circulating in plasma as aseries of multimers typically ranging in size from about 500 to 20,000kD (or 2 to 40 dimers of vWF). Dimers and multimeric forms of vWF arecomposed of 250 kD polypeptide subunits linked together by disulfidebonds. vWF mediates the initial platelet adhesion to the sub-endotheliumof the damaged vessel wall; only the larger multimers also exhibitinghemostatic activity. Multimerized VWF binds to the platelet surfaceglycoprotein Gp1bα, through an interaction in the Al domain of VWF, inorder to facilitate platelet adhesion. It is assumed that endothelialcells secret large polymeric forms of vWF and that those forms of vWFwhich have a low molecular weight (low molecular weight vWF) have arisenfrom proteolytic cleavage. The multimers having large molecular massesare stored in the Weibel-Palade bodies of the endothelial cells andliberated upon stimulation.

Reduction of FVIII binding activity, due to either reduced vWF proteinlevels or lowered FVIII binding affinity, results in one of three typesof von Willebrand's Disease. In addition to, or alternatively, certaintypes of von Willebrand's disease are characterized by an increase ordecrease in the level of Gp1bα-mediated platelet association, namely inTypes 2A, 2B, and 2M (summarized in Castaman et al., Disorders ofHemostasis 88(1):94-108 (2003)). As such, the modulation of vWFinteractions with both FVIII and Gp1bα is a viable strategy for thetreatment of both Haemophilia and von Willebrand's Disease.

Given the biological importance of vWF, there is a constant need in theart to improve ways for producing vWF for therapeutic applications. Itis well known that vWF can be isolated from endogenous sources, such ashuman blood plasma. The isolated vWF is advantageous in that it has ahigh specific activity for carrying out its biological function and can,therefore, be used effectively as a therapeutic protein for treatingrelated diseases, such as von Willebrand's disease. Typically, plasmavWF has a specific Ristocetin activity of about 100 mU/μg, but isolationfrom human blood plasma has disadvantages because, for example, theplasma can contain a variety of viruses, such as HIV and/or hepatitisviruses, which can be transferred to the patient. Furthermore, plasma isa limited resource and, thus, shortages of plasma can be problematic inproviding enough vWF for treatment. As such, recombinant methods forproducing vWF are advantageous in addressing some of the problemsassociated with relying on plasma as a source for vWF. For recombinantproduction, the full length of cDNA of vWF was cloned; thepropolypeptide corresponds to amino acid residues 23 to 764 of the fulllength prepro-vWF (Eikenboom et al (1995) Haemophilia 1, 77 90).

Unfortunately, vWF is a molecule with complex post-translationalmodifications. Also, the multimerization of the vWF dimers to large andultralarge multimers in the Golgi apparatus is a challenge forexpression in mammalian cells. For example, high molecular weight vWFexpressed in cell culture of, e.g., human (primary) endothelial cellsdepends on the specific storage of ultralarge vWF molecules inWeibel-Palade bodies. Such cell cultures are not suitable for theproduction of therapeutic proteins. Other cell culture methods have beenreported, and it is known that cell culture conditions can affect theproduction of vWF in a variety of ways. For instance, highconcentrations of ammonium (NH₄ ⁺) have been shown to disturbposttranslational modifications. Mayadas et al. (J. Biol. Chem.,264(23):13497-13503, 1989) demonstrated that levels of 25 mM ammoniumresulted in reduced vWF multimerization in endothethial cells, whichalso negatively affects the specific Ristocetin activity of recombinantvWF. Reduction of multimerization is generally associated in reductionof activity, particularly specific Ristocetin activity, of recombinantvWF.

It still remains difficult to predict which parameters can positively ornegatively affect production of a particular protein, especially complexglycoproteins like Factor VIII and vWF. For example, certain componentsof a cell culture medium have been shown to affect production of FactorVIII. As disclosed in U.S. Pat. No. 5,804,420, the addition of polyol,copper, and other trace metals can positively affect production yield ofFactor VIII. As also described in WO 2009/086309, cell culture processesusing copper in have been shown to improve production of Factor VIII.Expression of vWF in recombinant CHO cells has also been reported byMignot et al. (1989). However, none of these examples provideinformation regarding the specific activity of vWF or its multimericdistribution.

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin typeI motifs) proteins are a family of metalloproteinases containing anumber of conserved domains, including a zinc-dependant catalyticdomain, a cystein-rich domain, a disintegrin-like domain, and at leastone, and in most cases multiple, thrombospondin type I repeats (forreview, see Nicholson et al., BMC Evol Biol. 2005 Feb. 4; 5(1):11).These proteins, which are evolutionarily related to the ADAM and MMPfamilies of metalloproteinases (Jones G C, Curr Pharm Biotechnol. 2006February; 7(1):25-31), are secreted enzymes that have been linked to anumber of diseases and conditions including thrombotic thrombocytopenicpurpura (TTP) (Moake J L, Semin Hematol. 2004 January; 41(1):4-14),connective tissue disorders, cancers, inflammation (Nicholson et al.),and severe Plasmodium falciparum malaria (Larkin et al., PLoS Pathog.2009 March; 5(3):e1000349). Because of these associations, the ADAMTSenzymes have been recognized as potential therapeutic targets for anumber of pathologies (Jones G C, Curr Pharm Biotechnol. 2006 Febrary;7(1):25-31). Accordingly, methods of producing large yields of ADAMTSproteins having high specific activities, which are free of contaminantssuch as viruses, BSE, and pathogens like Mycoplasma bacteria, areneeded.

One ADAMTS family member, ADAMTS13, cleaves von Willebrand factor (vWF)between residues Tyr 1605 and Met 1606, a function responsible for thedegradation of large vWF multimers in vivo. Loss of ADAMTS13 activityhas been linked to a number of conditions, such as TTP (Moake J L, SeminHematol. 2004 January; 41(1):4-14), acute and chronic inflammation(Chauhan et al., J Exp Med. 2008 Sep. 1; 205(9):2065-74), and mostrecently, severe plasmodium falciparum malaria (Larkin et al., PLoSPathog. 2009 March; 5(3):e1000349).

The ADAMTS13 protease is a 190 kDa glycosylated protein producedpredominantly by the liver (Levy et al., Nature. 2001; 413:488-494;Fujikawa et al., Blood. 2001; 98:1662-1666; Zheng et al., J Biol Chem.2001; 276:41059-41063; Soejima et al., J Biochem (Tokyo). 2001;130:475-480; and Gerritsen et al., Blood. 2001; 98:1654-1661). Much likethe higher order rVWF multimers, recombinant expression of the largeADAMTS13 in mammalian cell culture presents many challenges.

Therefore, there is a need to provide cell culture conditions,particularly large-scale manufacturing culture conditions, that provideconsistent total protein yield and/or consistent total and specificactivity of the proteins produced between different cell cultures.Consistency among cultures in large-scale manufacturing processes is ofimportance in the manufacture of therapeutic proteins. There is also aneed for cell culture conditions for large-scale production of rVWF witha multimeric distribution and specific Ristocetin activity comparable orhigher than VWF as it is present in normal human plasma. Similarly, asADAMTS proteins have been implicated in a number of diseases andconditions, there is a need in the art for methods of large scaleproduction of recombinant ADAMTS proteins having high specificactivities, which are suitable for pharmaceutical formulation andadministration. The present invention satisfies these and other needs inthe art for the production of recombinant Von Willebrand Factor andrecombinant ADAMTS13.

BRIEF SUMMARY OF INVENTION

In certain aspects, the present invention is based on the surprisingfinding that supplementation of cell culture media used to expressrecombinant Von Willebrand Factor (rVWF) and recombinant ADAMTS13 (rA13)results in significantly improved protein expression and enzymaticactivity.

In a first aspect, the present invention provides a method for producinga recombinant Von Willebrand Factor (rVWF) composition, the methodcomprising the steps of: (a) providing a basal cell culture media; (b)supplementing the basal cell culture media with copper to provide afinal copper concentration of at least 2.4 μg/L; (c) providing one ormore cells comprising a nucleic acid encoding a rVWF protein; (d)culturing the one or more cells in the copper supplemented cell culturemedia such that rVWF is expressed and excreted from the cells into aculture supernatant; and (e) recovering at least a portion of theculture supernatant, wherein the recovered supernatant has a rVWFspecific ristocetin cofactor activity of at least 30 mU/μg rVWF.

In one embodiment of the methods provided above, the method furthercomprises a step of supplementing the basal cell culture media with ahydrolysate prior to culturing the one or more cells.

In one embodiment of the methods provided above, the hydrolysate is aplant hydrolysate. In a specific embodiment, the hydrolysate is a soyhydrolysate.

In one embodiment of the methods provided above, the basal cell culturemedia is an animal protein free culture media.

In one embodiment of the methods provided above, the basal cell culturemedia is a protein free culture media.

In one embodiment of the methods provided above, the basal cell culturemedia is a chemically defined culture media.

In one embodiment of the methods provided above, the final copperconcentration of the copper supplemented basal cell culture media is atleast 4 μg/L copper.

In one embodiment of the methods provided above, the final copperconcentration of the copper supplemented basal cell culture media isbetween 2.4 μg/L and 20 μg/L copper.

In one embodiment of the methods provided above, the coppersupplementing the basal cell culture media is provided as a copper salt,a copper chelate, or a combination thereof.

In one embodiment of the methods provided above, the copper salt isselected from the group consisting of copper sulfate, copper acetate,copper carbonate, copper chloride, copper hydroxide, copper nitrate, andcopper oxide.

In one embodiment of the methods provided above, the one or more cellsare mammalian cells. In a specific embodiment, the mammalian cells areCHO cells.

In one embodiment of the methods provided above, culturing the one ormore cells comprises batch cultivation of the cells.

In one embodiment of the methods provided above, culturing the one ormore cells comprises continuous cultivation of the cells. In a specificembodiment, the continuous cultivation of cells is performed inchemostatic mode. In another specific embodiment, the continuouscultivation of cells is performed in perfusion mode.

In one embodiment of the methods provided above, the one or more cellsis cultured in at least 100 L of the supplemented basal cell culturemedia.

In one embodiment of the methods provided above, the cell density ismaintained at less than 2.5×10⁶ cells per mL during the step ofculturing the one or more cells.

In one embodiment of the methods provided above, the cell density ismaintained at less than 2.0×10⁶ cells per mL during the step ofculturing the one or more cells.

In one embodiment of the methods provided above, the cell density ismaintained at less than 1.5×10⁶ cells per mL during the step ofculturing the one or more cells.

In one embodiment of the methods provided above, the step of recoveringat least a portion of the culture supernatant comprises filtration orcentrifugation to remove cells from the portion of culture supernatant.

In one embodiment of the methods provided above, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least40 mU/μg rVWF. In a specific embodiment, the recovered supernatant has arVWF specific ristocetin cofactor activity of at least 50 mU/μg rVWF. Ina more specific embodiment, the recovered supernatant has a rVWFspecific ristocetin cofactor activity of at least 60 mU/μg rVWF. In amore specific embodiment, the recovered supernatant has a rVWF specificristocetin cofactor activity of at least 70 mU/μg rVWF. In a yet morespecific embodiment, the recovered supernatant has a rVWF specificristocetin cofactor activity of at least 80 mU/μg rVWF.

In one embodiment of the methods provided above, at least 10% of therVWF in the supernatant is present in a high molecular weight VWFmultimer of more than 10 dimers. In a specific embodiment, at least 15%of the rVWF is present in a high molecular weight VWF multimer of morethan 10 dimers. In another specific embodiment, at least 20% of the rVWFis present in a high molecular weight VWF multimer of more than 10dimers. In another specific embodiment, at least 25% of the rVWF ispresent in a high molecular weight VWF multimer of more than 10 dimers.In another specific embodiment, at least 30% of the rVWF is present in ahigh molecular weight VWF multimer of more than 10 dimers.

In one embodiment of the methods provided above, the supernatantcontains high molecular weight VWF multimers of 14 to 22 dimers.

In one embodiment of the methods provided above, the NH₄ ⁺ content ofthe culture supernatant is maintained at a concentration below 10 mM.

In one embodiment of the methods provided above, the NH₄ ⁺ content ofthe culture supernatant is maintained at a concentration below 4 mM.

In one embodiment of the methods provided above, rVWF is co-expressedwith recombinant Factor VIII (rFVIII). In a specific embodiment, themethod further comprises a step of purifying rVWF away from at least 50%of the rFVIII present in the recovered supernatant. In one embodiment,the ratio of rVWF to rFVIII after the purification step is at least10:1.

In one embodiment of the methods provided above, the method furthercomprises a rVWF enrichment step.

In a second aspect, the present invention provides a recombinant VonWillebrand Factor (rVWF) composition prepared by a method providedherein.

In one embodiment of the compositions provided above, the compositionfurther comprises recombinant Factor VIII (rFVIII). In a specificembodiment, the ratio of rVWF to rFVIII is at least 10:1.

In one embodiment of the compositions provided above, the composition isformulated for pharmaceutical administration. In a specific embodiment,the composition is formulated for intravenous administration.

In a third aspect, the present invention provides a cell culturesupernatant comprising recombinant Von Willebrand Factor (rVWF), whereinthe supernatant is prepared by a method provided herein.

In a fourth aspect, the present invention provides a cell culturesupernatant comprising recombinant Von Willebrand Factor (rVWF), whereinat least 10% of the rVWF in the supernatant is present in a highmolecular weight VWF multimer of more than 10 dimers. In a specificembodiment, at least 15% of the rVWF in the supernatant is present in ahigh molecular weight VWF multimer of more than 10 dimers. In anotherspecific embodiment, at least 20% of the rVWF in the supernatant ispresent in a high molecular weight VWF multimer of more than 10 dimers.In another specific embodiment, at least 25% of the rVWF in thesupernatant is present in a high molecular weight VWF multimer of morethan 10 dimers. In another specific embodiment, at least 30% of the rVWFin the supernatant is present in a high molecular weight VWF multimer ofmore than 10 dimers. In yet another specific embodiment of thesupernatants provided above, the supernatant is prepared according to amethod provided herein.

In a fifth aspect, the present invention provides a cell culturesupernatant comprising recombinant Von Willebrand Factor (rVWF), whereinthe supernatant contains at least 0.4 IU ristocetin cofactor activityper mL. In a specific embodiment, the supernatant contains at least 0.5IU ristocetin cofactor activity per mL. In another specific embodiment,the supernatant contains at least 0.6 IU ristocetin cofactor activityper mL. In another specific embodiment, the supernatant contains atleast 0.7 IU ristocetin cofactor activity per mL. In yet anotherspecific embodiment of the supernatants provided above, the supernatantis prepared according to a method provided herein.

In a sixth aspect, the present invention provides a method for producinga recombinant ADAMTS13 (rA13) composition, the method comprising thesteps of: (a) providing a basal cell culture media; (b) supplementingthe basal cell culture media with copper to provide a final copperconcentration of at least 1.0 μg/L; (c) providing one or more cellscomprising a nucleic acid encoding a rA13 protein; (d) culturing the oneor more cells in the copper supplemented cell culture media such thatrA13 is expressed and excreted from the cells into a culturesupernatant; and (e) recovering at least a portion of the culturesupernatant, wherein at least 1500 Units FRETS-VWF73 activity per litersupplemented basal cell culture media per day is present in therecovered culture supernatant.

In one embodiment of the methods provided above, the basal cell culturemedia is an animal protein free culture media.

In one embodiment of the methods provided above, the basal cell culturemedia is a protein free culture media.

In one embodiment of the methods provided above, the basal cell culturemedia is a chemically defined culture media.

In one embodiment of the methods provided above, the final copperconcentration of the supplemented basal cell culture media is at least 1μg/L copper.

In one embodiment of the methods provided above, the final copperconcentration of the supplemented basal cell culture media is at least 2μg/L copper.

In one embodiment of the methods provided above, the final copperconcentration of the supplemented basal cell culture media is at least 4μg/L copper.

In one embodiment of the methods provided above, the final copperconcentration of the supplemented basal cell culture media is between 1μg/L and 6 μg/L copper.

In one embodiment of the methods provided above, the final copperconcentration of the supplemented basal cell culture media is between 2μg/L and 4 μg/L copper.

In one embodiment of the methods provided above, copper supplementingthe basal cell culture media is provided as a copper salt, a copperchelate, or a combination thereof. In a specific embodiment, the coppersalt is selected from the group consisting of copper sulfate, copperacetate, copper carbonate, copper chloride, copper hydroxide, coppernitrate, and copper oxide.

In one embodiment of the methods provided above, the one or more cellsare mammalian cells. In a specific embodiment, the mammalian cells areCHO cells.

In one embodiment of the methods provided above, culturing the one ormore cells comprises batch cultivation of the cells.

In one embodiment of the methods provided above, culturing the one ormore cells comprises continuous cultivation of the cells. In a specificembodiment, the continuous cultivation of cells is performed inchemostatic mode. In another specific embodiment, the continuouscultivation of cells is performed in perfusion mode.

In one embodiment of the methods provided above, the one or more cellsis cultured in at least 100 L of the supplemented basal cell culturemedia.

In one embodiment of the methods provided above, the cell density ismaintained at less than 4.0×10⁶ cells per mL during the step ofculturing the one or more cells.

In one embodiment of the methods provided above, the cell density ismaintained at less than 3.5×10⁶ cells per mL during the step ofculturing the one or more cells.

In one embodiment of the methods provided above, the cell density ismaintained at less than 3.0×10⁶ cells per mL during the step ofculturing the one or more cells.

In one embodiment of the methods provided above, the cell density ismaintained at less than 2.5×10⁶ cells per mL during the step ofculturing the one or more cells.

In one embodiment of the methods provided above, the cell density ismaintained at less than 2.0×10⁶ cells per mL during the step ofculturing the one or more cells.

In one embodiment of the methods provided above, the cell density ismaintained at less than 1.5×10⁶ cells per mL during the step ofculturing the one or more cells.

In one embodiment of the methods provided above, the step of recoveringat least a portion of the culture supernatant comprises filtration orcentrifugation to remove cells from the portion of culture supernatant.

In one embodiment of the methods provided above, at least 2000 UnitsFRETS-VWF73 activity per liter supplemented basal cell culture media perday is present in the recovered culture supernatant.

In one embodiment of the methods provided above, at least 2500 UnitsFRETS-VWF73 activity per liter supplemented basal cell culture media perday is present in the recovered culture supernatant.

In one embodiment of the methods provided above, the recoveredsupernatant has a rA13 specific FRETS-VWF73 activity of at least 800mU/μg.

In a preferred embodiment of the methods provided above, the recoveredsupernatant has a rA13 specific FRETS-VWF73 activity of at least 1200mU/μg.

In a more preferred embodiment of the methods provided above, therecovered supernatant has a rA13 specific FRETS-VWF73 activity of atleast 1600 mU/μg.

In one embodiment of the methods provided above, the NH₄ ⁺ content ofthe culture supernatant is maintained at a concentration below 10 mM.

In one embodiment of the methods provided above, the NH₄ ⁺ content ofthe culture supernatant is maintained at a concentration below 5 mM.

In one embodiment of the methods provided above, the NH₄ ⁺ content ofthe culture supernatant is maintained at a concentration below 4 mM.

In one embodiment of the methods provided above, the method furthercomprises a rA13 enrichment step.

In a seventh aspect, the present invention provides a cell culturesupernatant comprising recombinant ADAMTS13 (rA13), wherein thesupernatant is prepared by a method provided herein.

In an eighth aspect, the present invention provides a cell culturesupernatant comprising recombinant ADAMTS13 (rA13), wherein thesupernatant contains at least 5 U FRETS-VWF73 activity per mL. In aspecific embodiment, the supernatant contains at least 6 U FRETS-VWF73activity per mL. In another specific embodiment, the supernatantcontains at least 7 U FRETS-VWF73 activity per mL. In another specificembodiment, the supernatant contains at least 8 U FRETS-VWF73 activityper mL. In another specific embodiment, the supernatant contains atleast 9 U FRETS-VWF73 activity per mL. In another specific embodiment,the supernatant contains at least 10 U FRETS-VWF73 activity per mL. Inyet another specific embodiment of the supernatants provided above, thesupernatant is prepared according to a method provided herein.

In a ninth aspect, the present invention provides a cell culturesupernatant comprising recombinant ADAMTS13 (rA13), wherein thesupernatant contains at least 2 μg rA13 per mL. In a specificembodiment, the supernatant contains at least 3 μg rA13 per mL. Inanother specific embodiment, the supernatant contains at least 4 μg rA13per mL. In another specific embodiment, the supernatant contains atleast 5 μg rA13 per mL. In another specific embodiment, the supernatantcontains at least 6 μg rA13 per mL. In yet another specific embodimentof the supernatants provided above, the supernatant is preparedaccording to a method provided herein.

In a tenth aspect, the present invention provides a recombinant ADAMTS13(rA13) composition prepared by a method according to any one of themethods described above.

In one embodiment of the compositions provided above, the composition isformulated for pharmaceutical administration. In a specific embodiment,the composition is formulated for intravenous administration.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. (1A) Low resolution (1%) agarose gel electrophoresis of rVWFexpressed in mammalian cell culture in the presence of low (1.0 μg/L)and high (4.3 μg/L) copper concentration, as described in Example 2.Note that culture Day 3 is equivalent to batch day 1 of Table 7 andTable 8. (1B) The relative amounts of VWF multimers having 1 to 10dimers (band number 1) and more than 10 dimers (band number 2), asindicated by the bands defined in FIG. 1A, was quantitated bydensitometric analysis.

FIG. 2. (2A) Interval plot of the average rVWF specific activity presentin rVWF cell culture supernatants grown at high and low cell densitiesin the presence of high or low levels of copper. (2B) Interval plot ofthe average NH₄ ⁺ concentration found in rVWF cell culture supernatantsgrown at high and low cell densities in the presence of high or lowlevels of copper.

FIG. 3. Supernatants from cell cultures expressing recombinant ADAMTS13in the presence of increasing levels of copper were investigated bySDS-PAGE analysis. rA13 was visualized after SDS-PAGE by (3A) silverstaining and (3B) anti-A13 western blotting.

FIG. 4. Plot of volumetric productivity (P Frets) data versus copperconcentration showing an extrapolation (solid line) of optimal copperconcentration effect on rA13 productivity.

FIG. 5A-K. Bar graphs from continuous suspension (chemostat) cellcultures expressing rA13 over a culture time of 8 weeks comparing theeffects of basal levels of copper (0.66 μg/L) to that of culturessupplemented to a final concentration of 2 μg/L copper. Each barrepresents the mean data of one week of chemostatic culturing. Thelegend refers to the particular week represented in the data.

FIG. 6. (6A) Low resolution (1%) agarose gel electrophoresis of rVWFexpressed in mammalian cell culture in the presence of low (1.0 μg/L)and high (4.3 μg/L) copper concentration under high and low celldensities as described in Example 3. Note that culture Day 8 and 17(“CST8” and “CST17”) is equivalent to Day 8 and Day 17 of Table 10 toTable 13. (6B) The relative amounts of VWF multimers having 1 to 10dimers and more than 10 dimers, as indicated by the bands defined inFIG. 6A, was quantitated by densitometric analysis.

DETAILED DESCRIPTION OF INVENTION I. INTRODUCTION

Recombinant vWF (rVWF) and recombinant ADAMTS13 (rA13) can be producedby expression in large-scale mammalian cell cultures. However, theactivity of these proteins when produced using standard cell cultureconditions often varies across cell cultures, even when the generalformulations of the media are not changed, and the specific activitiesof the recombinant proteins are often not equal to that of vWF and rA13derived from blood plasma. In addition, rVWF expressed in mammalian cellcultures tends to produce protein compositions with low (under 10%)percentages of higher order multimers (higher order multimers includemolecules containing more than 10 VWF dimers). These drawbacks instandard production methods of rVWF and rA13 are particularlyproblematic when developing cultures for large scale production (i.e.,from 10 to over 20,000 liter cultures).

One potential source of the variability often seen in different cellculture batches is the presence of contaminants in components of thecell culture media. These contaminants may be present in differentamounts in different batches, leading to variable results in theproduction of rVWF and rA13. After investigating the differentcontaminants found in various cell culture media additives, the presentinventors found that the presence of hydrolysates leads to variation incopper concentrations in the cell media. Further investigation providedthe surprising result that supplementing copper concentrations in cellmedia to produce a total copper concentration of at least about 1 μg/Lto about 20 μg/L consistently increased the total and specific activityof rVWF and rA13 and/or could also lead to increased total proteinyield. Thus, the present invention provides methods and compositions forhigh yield production of rVWF and rA13 proteins with high specificactivity.

In one aspect, the present invention provides cell culture methods andcompositions for producing large quantities of rVWF and rA13 withactivity that is comparable to or higher than that seen with plasmaderived vWF (pdVWF) or plasma derived ADAMTS13 (pdA13). In furtheraspects, the rVWF and rA13 proteins produced in accordance with thepresent invention show consistently higher activity than proteinsproduced using standard cell culture methods in media that has not beensupplemented with copper or other supplements described in furtherdetail herein. Advantageously, in certain embodiments of the methods andcompositions provided herein, the rVWF and rA13 proteins produced inaccordance with the present invention show consistently higher specificactivity (i.e., U/mg protein) than proteins produced using standard cellculture methods in media that has not been supplemented with copper orother supplements described in further detail herein. Likewise, themethods provided herein for the production of rVWF and rA13 providehigher yields of activity per volume culture (i.e., U/L/D), as comparedto standard cell culture methods utilizing media that has not beensupplemented with copper or other supplements described in furtherdetail herein.

In a further aspect, the present invention provides cell culture methodsin which a basal cell culture medium is supplemented with copper toresult in a total concentration of at least about 1 μg/L. In otherembodiments, the basal cell culture medium is supplemented with copperto result in a total concentration of at least about 2 μg/L. In yetother embodiments, the basal cell culture medium is supplemented withcopper to result in a total concentration of at least about 1 μg/L toabout 20 μg/L. In some embodiments, the total concentration of copper isfrom about 1.5-4.5 μg/L. In certain embodiments, the cell culture mediumis supplemented to result in about 1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4,2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5.0, 5.5, 6.0,6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20 μg/L copper, or more. Basal cell culture media generally have atrace copper concentration of under 1 μg/L.

In some embodiments, the present invention provides cell culture methodsin which a basal cell culture medium is supplemented with from about 1.0to about 20 μg/L copper for production of rVWF. In further embodiments,the basal cell culture medium is supplemented with from about 1.5-15,2.0-10, 2.5-8, 3.0-6, 4.0 -5.0 μg/L copper for production of rVWF. Instill further embodiments, the basal cell culture medium may, inaddition to the supplemented copper, also include one or morehydrolysates.

In other embodiments, the present invention provides cell culturemethods in which a basal cell culture medium is supplemented with fromabout 1.5 to about 4 μg/L copper for production of rA13. In furtherembodiments, the basal cell culture medium is supplemented with fromabout 1.6-3.8, 1.7-3.6, 1.8-3.4, 1.9-3.2, 2.0-3.0, 2.1-2.8, 2.2-2.6,2.3-2.4 μg/L copper for production of rA13. In still furtherembodiments, the basal cell culture medium may, in addition to thesupplemented copper, also include one or more hydrolysates. In yetfurther embodiments, the basal cell culture medium includes, in additionto copper and/or one or more hydrolysates, about 1.0 to about 30 μMzinc. In still further embodiments, the basal cell culture mediumfurther includes, in addition to copper and/or one or more hydrolysatesand/or zinc, between about 0.5 to about 5.0 mM calcium.

In a still further aspect and in accordance with any of the above, thepresent invention provides cell culture methods in which the ammoniumlevels of the cell culture solution are low (under 10 mM). In certainembodiments, cell culture methods of the present invention utilize cellculture media having over 1, 2, 3, 4, or 5 μg/L copper in combinationwith low levels of ammonium.

One of the advantages of the methods and compositions of the presentinvention is that they are amenable to large scale cell cultureproduction. These large scale cell cultures are at least 10 L, 50 L, 100L, 150 L, 200 L, 250 L, 500 L, 750 L, 1,000 L, 1,500 L, 2,000 L, 5,000L, 10,000 L, or 20,000 liter cultures.

In certain aspects, the methods of the invention do not necessarilyresult in a higher amount of recombinant protein overall, but therecombinant protein (either rVWF or rA13) that is produced shows highertotal and specific activity than is seen in proteins produced usingstandard cell cultures, particularly as compared to proteins produced incell cultures in which the cell culture medium has not been supplementedwith additional copper. In further aspects, the rVWF and rA13 proteinsproduced in cells cultured in copper supplemented media showconsistently increased activity per liter of cell culture as compared tocells cultured in basal cell culture media that has not beensupplemented with copper. In still further aspects, the coppersupplemented media of the present invention result in increased proteinyield, increased number of cells in the culture, and/or increased totalactivity per liter of culture as compared to media that has not beensupplemented with copper.

Still further advantages of methods and compositions of the invention isthat they result a population of proteins containing a high percentage(over 10%) of highly multimerized rVWF.

Although much of the discussion herein regarding ADAMTS proteins is interms of ADAMTS13 (A13), it will be appreciated that because all ADAMTSproteins share a common core domain architecture and commonstructure-function relationships, the methods and compositions describedherein are applicable for production of any ADAMTS proteins, not onlyrA13.

II. DEFINITIONS

As used herein, “recombinant vWF” includes vWF obtained via recombinantDNA technology. In certain embodiments, vWF proteins of the inventioncan comprise a construct, for example, prepared as in WO 1986/06096published on Oct. 23, 1986 and U.S. patent application Ser. No.07/559,509, filed on Jul. 23, 1990, in the name of Ginsburg et al.,which is incorporated herein by reference with respect to the methods ofproducing recombinant vWF. The vWF in the present invention can includeall potential forms, including the monomeric and multimeric forms. Itshould also be understood that the present invention encompassesdifferent forms of vWF to be used in combination. For example, the vWFof the present invention may include different multimers, differentderivatives and both biologically active derivatives and derivatives notbiologically active.

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

In the context of the present invention, the recombinant vWF embracesany member of the vWF family from, for example, a mammal such as aprimate, human, monkey, rabbit, pig, rodent, mouse, rat, hamster,gerbil, canine, feline, and biologically active derivatives thereof. Ina preferred embodiment, the recombinant VWF is human VWF. Mutant andvariant vWF proteins having activity are also embraced, as arefunctional fragments and fusion proteins of the vWF proteins.Furthermore, the vWF of the invention may further comprise tags thatfacilitate purification, detection, or both. The vWF described hereinmay further be modified with a therapeutic moiety or a moiety suitableimaging in vitro or in vivo.

The terms “highly multimeric vWF,” “high molecular weight vWF,” and “HMWVWF” may be used interchangeably and refer to covalently attached vWFmultimers containing more than 10 VWF dimers. In certain embodiments,HMW VWF contains at least 11 VWF dimers, or at least 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, or more VWF dimers.

As used herein, an “ADAMTS protein” refers to a polypeptide of thedisintegrin and metalloproteinase with thrombospondin type I motifsfamily of metalloproteinases. Members of this family include the humanproteins ADAMTS1 (NM_006988), ADAMTS2 (NM_014244; NM_021599), ADAMTS3(NM_014243), ADAMTS4 (NM_005099), ADAMTS5 (NM_007038), ADAMTS6(NM_014273), ADAMTS7 (NM_0142727), ADAMTS8 (NM_007037), ADAMTS9(NM_182920; NM_182921; NM_020249), ADAMTS10 (NM_030957), ADAMTS12(NM_030955), ADAMTS13 (NM_139025; NM_139026; NM_139027; NM_139028),ADAMTS14 (NM_139155; NM_080722), ADAMTS15 (NM_139055), ADAMTS16(NM_139056), ADAMTS17 (NM_139057), ADAMTS18 (NM_199355; NM_139054),ADAMTS19 (NM_133638), and ADAMTS20 (NM_025003, NM_175851). ADAMTSproteins include both full-length proteins and partial polypeptides thatdisplay at least partial biological activity, for example, at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the activitydemonstrated by the full-length protein, in particular the proteaseactivity demonstrated by the full length protein. In certain instances,an ADAMTS protein will be post-translationally modified either in vivoor in vitro, for example, by enzymatic or chemical means. It isunderstood that the ADAMTS proteins of the present invention includealternatively spliced isoforms, conservatively modified proteins,substantially identical proteins, homologues, and the like.

In the context of the present invention, an ADAMTS protein embraces anymember of the ADAMTS family from, for example, a mammal such as aprimate, human, monkey, rabbit, pig, rodent, mouse, rat, hamster,gerbil, canine, feline, and biologically active derivatives thereof.Mutant and variant ADAMTS proteins having activity are also embraced, asare functional fragments and fusion proteins of the ADAMTS proteins.Furthermore, the ADAMTS proteins of the invention may further comprisetags that facilitate purification, detection, or both. The ADAMTSproteins described herein may further be modified with a therapeuticmoiety or a moiety suitable imaging in vitro or in vivo.

As used herein, an “ADAMTS13 protein” refers to any protein orpolypeptide with ADAMTS13 activity, particularly the ability to cleavethe peptide bond between residues Tyr-842 and Met-843 of VWF. In anexemplary embodiment, an ADAMTS13 protein refers to a polypeptidecomprising an amino acid sequence that is highly similar to that ofNP_620594 (ADAMTS13 isoform 1, preproprotein) or amino acids 75 to 1427of NP_620594 (ADAMTS13 isoform 1, mature polypeptide). In anotherembodiment, an ADAMTS13 protein refers to a polypeptide comprising anamino acid sequence that is highly similar to that of NP_620596(ADAMTS13 isoform 2, preproprotein) or amino acids 75 to 1371 ofNP_620594 (ADAMTS13 isoform 2, mature polypeptide). In yet anotherembodiment, ADAMTS13 proteins include polypeptides comprising an aminoacid sequence highly similar to that of NP_620595 (ADAMTS13 isoform 3,preproprotein) or amino acids 75 to 1340 of NP_620595 (ADAMTS13 isoform1, mature polypeptide). As used herein, an ADAMTS13 protein includesnatural variants with vWF cleaving activity and artificial constructswith vWF cleaving activity. As used in the present invention, ADAMTS13encompasses any natural variants, alternative sequences, isoforms ormutant proteins that retain some basal activity. Examples of ADAMTS13mutations found in the human population include, without limitation,R7W, V88M, H96D, R102C, R193W, T196I, H234Q, A250V, R268P, W390C, R398H,Q448E, Q456H, P457L, C508Y, R528G, P618A, R625H, I673F, R692C, A732V,S903L, C908Y, C951G, G982R, C1024G, A1033T, R1095W, R1123C, C1213Y,T12261, G1239V, R1336W, many of which have been found associated withthrombotic thrombocytopenic purpura (TTP). ADAMTS13 proteins alsoincludes polypeptides containing post-translational modifications. Forexample, ADAMTS13 has been shown to be modified by N-acetylglucosamine(GlcNAc) at residues 614, 667, and 1354, and it has been predicted thatresidues 142, 146, 552, 579, 707, 828, and 1235 may also be modified inthis fashion.

Proteolytically active recombinant ADAMTS13 may be prepared byexpression in mammalian cell cultures, as described in Plaimauer et al.,(2002, Blood. 15; 100(10):3626-32) and US 2005/0266528, the disclosuresof which are herein incorporated by reference in their entireties forall purposes. Methods for the expression of recombinant ADAMTS13 in cellculture are disclosed in Plaimauer B, Scheiflinger F. (Semin Hematol.2004 January; 41(1):24-33 and US 2011/0086413, the disclosures of whichare herein incorporated by reference in their entireties for allpurposes.

As used herein, the term “biologically active derivative”, when used inthe context of an ADAMTS protein, also embraces polypeptides obtainedvia recombinant DNA technology. This may include any method known in theart for (i) the production of recombinant DNA by genetic engineering,e.g., via reverse transcription of RNA and/or amplification of DNA, (ii)introducing recombinant DNA into prokaryotic or eukaryotic cells bytransfection, i.e., via electroporation or microinjection, (iii)cultivating said transformed cells, e.g., in a continuous or batch-wisemanner, (iv) expressing an ADAMTS protein, e.g., constitutively or uponinduction, and (v) isolating said ADAMTS protein, e.g., from the culturemedium or by harvesting the transformed cells, in order to (vi) obtainsubstantially purified recombinant ADAMTS protein, e.g., via ionexchange chromatography, size exclusion chromatography, affinitychromatography, hydrophobic interaction chromatography, and the like.The term “biologically active derivative” includes also chimericmolecules such as e.g. an ADAMTS protein, or functional fragmentthereof, in combination with a second polypeptide, e.g., animmunoglobulin Fc domain or an albumin domain, in order to improve thebiological/pharmacological properties such as e.g., half life of theADAMTS protein in the circulation system of a mammal, particularly ahuman.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. In one embodiment, rVWF is thepredominant species present in a preparation is substantially purified.In another embodiment, rA13 is the predominant species present in apreparation is substantially purified. The term “purified” in someembodiments denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. In other embodiments, itmeans that the nucleic acid or protein is at least 50% pure, morepreferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or more pure. “Purify” or “purification” in other embodimentsmeans removing at least one contaminant from the composition to bepurified. In this sense, purification does not require that the purifiedcompound be homogenous, e.g., 100% pure.

The biological activity of vWF can be measured by known in vitro assays.For example, the Ristocetin Cofactor assay is based on the agglutinationof fresh or formalin-fixed platelets induced by the antibioticristocetin in the presence of vWF. The degree of platelet agglutinationdepends on the vWF concentration and can be measured by theturbidimetric method, e.g. by use of an aggregometer (Weiss et al., J.Clin. Invest. 52: 2708-2716, 1973; Macfarlane et al., Thromb. Diath.Haemorrh. 34: 306-308, 1975). As provided herein, the specificRistocetin Cofactor activity of the vWF of the present invention isdescribed in terms of mU/μg of vWF, as measured using in vitro assays.

As used herein, “one unit of ADAMTS activity” is defined as the amountof activity in 1 mL of pooled normal human plasma, regardless of theassay being used. For example, when the ADAMTS protein is ADAMTS13, oneunit of ADAMTS13 FRETS-VWF73 activity is the amount of activity neededto cleave the same amount of FRETS-VWF73 substrate (Kokame et al., Br JHaematol. 2005 April; 129(1):93-100) as is cleaved by one mL of poolednormal human plasma. Conveniently, ADAMTS13 activity may be determinedby functional assays, such as functional assays employing modified vonWillebrand factor peptides as substrate for ADAMTS13 (Tripodi et al. JThromb Haemost. 2008 September; 6(9): 1534-41). A preferred method ofdetermining recombinant human ADAMTS13 activity is disclosed inGerritsen et al. (Assay of von Willebrand factor (vWF)-cleaving proteasebased on decreased collagen binding affinity of degraded vWF: a tool forthe diagnosis of thrombotic thrombocytopenic purpura (TTP). ThrombHaemost 1999; 82: 1386-1389). In one embodiment, to be considered as aADAMTS13 protein as defined above, a polypeptide or protein must have atleast 1% of the vWF cleaving activity of native ADAMTS13. In otherembodiments, an ADAMTS13 protein will contain at least 10% of theactivity of native ADAMTS13. In yet other embodiments, an ADAMTS13protein will contain at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or 100% of the activity of native ADAMTS13. The quantity of anADAMTS13 protein may also be determined by measurement of an ADAMTS13antigen, for example using the ELISA method disclosed in Rieger et al.,(2006, Thromb Haemost. 2006 95(2):212-20). A well established conventionin the art is that 1 mL of pooled normal human plasma contains 1 μg ofADAMTS13. Thus, the convention in the art is that 1 μg of plasma-derivedADAMTS13 has one unit of ADAMTS13 activity.

The terms “cell culture solution,” “cell culture medium or media,” and“cell culture supernatant” refer to aspects of cell culture processesgenerally well known in the art. In the context of the presentinvention, a cell culture solution can include cell culture media andcell culture supernatant. The cell culture media are externally added tothe cell culture solution, optionally together with supplements, toprovide nutrients and other components for culturing cells expressingrVWF or rA13. The cell culture supernatant refers to a cell culturesolution comprising the nutrients and other components from the cellculture medium as well as products released, metabolized, and/orexcreted from the cells during culture, but not the cells themselves.Thus in one context, a cell culture supernatant can refer to the liquidphase of a cell culture solution (i.e., the cell culture solutionexcluding the cells). For example, the ammonium concentration of aculture supernatant generally refers to the ammonium concentrationpresent in the cell culture solution. In other contexts, a cell culturesupernatant refers to a cell culture solution from which the cells havebeen removed (i.e., a recovered cell culture supernatant).

As used herein, the terms “vitamin B3,” “nicotinamide,” “niacinamide,”“niacin,” and “nicotinic acid” may be used interchangeably to refer toany member of the B3 family of vitamins. Accordingly, any member of thisfamily may be used to supplement medium used in the methods of thepresent invention.

As used herein, the term “chemically defined medium” or “chemicallydefined media” refers to a synthetic growth medium in which the identityand concentration of all the components are known. Chemically definedmediums do not contain bacterial, yeast, animal, or plant extracts,although they may or may not include individual plant or animal-derivedcomponents (e.g., proteins, polypeptides, etc.). Non-limiting examplesof commercially available chemically defined mediums include, variousDulbecco's Modified Eagle's (DME) mediums (Sigma-Aldrich Co; SAFCBiosciences, Inc), Ham's Nutrient Mixture (Sigma-Aldrich Co; SAFCBiosciences, Inc), combinations thereof, and the like. Methods ofpreparing chemically defined culture mediums are known in the art, forexample in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, andU.S. Patent Application Publication Numbers 2008/0009040 and2007/0212770, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

As used herein, the term “oligopeptide-free culture medium” or“oligopeptide-free culture media” refers to a protein-free medium thatdoes not comprise oligopeptides, such as, e.g., oligopeptides derivedfrom a protein hydrolysate. In one embodiment, the medium does notcomprise oligopeptides having twenty or more amino acids. In oneembodiment of the present invention, the medium does not compriseoligopeptides having fifteen or more amino acids. In another embodimentof the invention, the medium does not comprise oligopeptides having tenor more amino acids. In one embodiment the medium does not compriseoligopeptides having seven or more amino acids. In another embodimentthe medium does not comprise oligopeptides having five or more aminoacids. In still another embodiment the medium does not compriseoligopeptides having three or more amino acids. According to a furtherembodiment of the present invention, the medium does not compriseoligopeptides having two or more amino acids. Methods of preparingoligopeptide-free culture medium are known in the art, for example inU.S. Pat. Nos. 6,171,825 and 6,936,441, WO 2007/077217, and U.S. PatentApplication Publication Numbers 2008/0009040 and 2007/0212770, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

As used herein, the term “serum-free culture medium” or “serum-freeculture media” refers to a culture medium that is not supplemented withan animal serum. Although oftentimes serum-free mediums are chemicallydefined mediums, serum-free mediums may be supplemented with discreteanimal or plant proteins or protein fractions. Methods of preparingserum-free culture medium are known in the art, for example in U.S. Pat.Nos. 6,171,825 and 6,936,441, WO 2007/077217, and U.S. PatentApplication Publication Numbers 2008/0009040 and 2007/0212770, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

As used herein, the term “animal protein-free culture medium” or “animalprotein-free culture media” refers to a culture medium that is notsupplemented with an animal serum, protein, or protein fraction.Although oftentimes animal protein-free culture mediums are chemicallydefined mediums, animal protein-free culture mediums may contain plantor yeast hydrolysates. Methods of preparing animal protein-free culturemedium are known in the art, for example in U.S. Pat. Nos. 6,171,825 and6,936,441, WO 2007/077217, and U.S. Patent Application PublicationNumbers 2008/0009040 and 2007/0212770, the disclosures of which areincorporated herein by reference in their entireties for all purposes.

As used herein, the term “basal cell culture medium” or “basal cellculture media” refers to a chemically defined culture medium, anoligopeptide-free culture medium, a serum-free culture medium, or ananimal protein-free culture medium that has not been supplemented with ahydrolysate, e.g., a plant or yeast hydrolysate. basal mediums are wellknown in the art, e.g., DMEM, Ham's F12, DMEM/Ham's F12, Medium 199,McCoy, or RPMI. The basal medium can include a number of ingredients,including amino acids, vitamins, organic and inorganic salts, andsources of carbohydrate. Each ingredient can be present in an amountthat supports the cultivation of a cell, such amounts being generallyknown to a person skilled in the art. The medium can include auxiliarysubstances, such as buffer substances, e.g., sodium bicarbonate,antioxidants, stabilizers to counteract mechanical stress, or proteaseinhibitors. If necessary, a non-ionic surfactant such as copolymersand/or mixtures of polyethylene glycols and polypropylene glycols can beadded.

III. CELL CULTURE MEDIA AND CELL CULTURE SUPERNATANT

One aspect of the present invention relates to cell culture media forproducing rVWF and/or rA13 that have increased activity as compared torVWF and rA13 produced with basal cell culture media. In one aspect, thepresent invention relates to cell culture media for producing rVWFand/or rA13 in which basal cell culture media is supplemented with oneor more additional substances. In specific embodiments and as isdiscussed in further detail below, cell culture conditions of thepresent invention include basal cell culture media supplemented to haveat least 1.0 μg/L copper. In further embodiments, cell culture media ofuse and supernatants derived from the processes in the present inventionalso comprise low levels (under 10 mM) of ammonium. In a particularembodiment, the cell culture conditions used for expressing rVWF and/orrA13 are controlled such that the cell culture supernatant maintains alow level of ammonium, i.e., less than 10 mM and preferably less than 5mM.

The culture media of the present invention can be based on a suitablebasal medium well known in the art, e.g., DMEM, Ham's F12, DMEM/Ham'sF12, Medium 199, McCoy, or RPMI. The basal medium can include a numberof ingredients, including amino acids, vitamins, organic and inorganicsalts, and sources of carbohydrate. Each ingredient can be present in anamount that supports the cultivation of a cell, such amounts beinggenerally known to a person skilled in the art. The medium can includeauxiliary substances, such as buffer substances, e.g., sodiumbicarbonate, antioxidants, stabilizers to counteract mechanical stress,or protease inhibitors. If necessary, a non-ionic surfactant such ascopolymers and/or mixtures of polyethylene glycols and polypropyleneglycols can be added.

Generally, basal media contain less than 1 μg/L of copper—for example,DMEM/Ham's F12 has a copper concentration of about 0.3 μg/L. Suchconcentrations of copper do not provide enough copper ions to supportproduction of rVWF and rA13 proteins of the present invention, whichshow high specific activity.

Copper can be provided to cell culture media of the present inventionthrough a variety of ways, such as through addition of a mediumsupplement. In some embodiments, the medium supplement can containhydrolysate, which can be provided to increase the copper concentrationin the media. Hydrolysates can include any hydrolysate well known in theart, such as plant hydrolysates, soy hydrolysates, and wheat glutenhydrolysate. In certain embodiments, addition of hydrolysate cancontribute an increased copper concentration of about 0.2 to about 10μg/L of Cu²⁺. In some embodiments, the amount of copper provided by thehydrolysate can depend on the amount of copper in the hydrolysate aswell as the amount of hydrolysate added. The copper content of ahydrolysate can be determined by elemental analysis, e.g., atomadsorption spectroscopy (GFAA: graphite furnace atomic adsorption), ormass spectroscopy methods (e.g., ICP-MS).

In certain embodiments, copper can be provided to the culture mediaalone or in addition to hydrolysate by providing a medium supplementincluding a suitable copper salt or copper chelate. Suitable coppersalts can include but are not limited to copper sulfate, copper acetate,copper carbonate, copper chloride, copper hydroxide, copper nitrate, andcopper oxide. Suitable copper chelators can include but are not limitedto albumin, ethylenediaminetetraacetic acid (EDTA), polyamine chelatingagents, ethylenediamine, diethylenetriamine, triethylenetetramine,triethylenediamine, tetraethylenepentamine, aminoethylethanolamine,aminoethylpiperazine, pentaethylenehexamine,triethylenetetramine-hydrochloride,tetraethylenepentamine-hydrochloride,pentaethylenehexamine-hydrochloride, tetraethylpentamine, captopril,penicilamine, N,N′-bis(3-aminopropyl)-1,3-propanediamine, N,N,Bis (2animoethyl) 1,3 propane diamine, 1,7-dioxa-4,10-diazacyclododecane,1,4,8,11-tetraaza cyclotetradecane-5,7-dione, 1,4,7-triazacyclononanetrihydrochloride, 1-oxa-4,7,10-triazacyclododecane, 1,4,8,12-tetraazacyclopentadecane, and 1,4,7,10-tetraaza cyclododecane.

In certain embodiments, basal cell culture media is supplemented withcopper to result in a total copper concentration of about 1.0 to about20 μg/L. In a specific embodiment, the basal cell media is supplementedwith copper to a final concentration of between about 1.0 to about 10μg/L. In further embodiments, the basal cell culture media issupplemented to result in a final copper concentration of about 1.0-5.0,1.2-4.0, 1.3-3.0, 1.4-2.9, 1.5-2.8, 1.6-2.7, 1.7-2.6, 1.8-2.5, 1.9-2.4,2.0-2.3, 2.1-2.2 μg/L copper. In further embodiments, the basal cellculture media used in methods of the present invention are supplementedto result in about 1.2-9.5, 1.4-9, 1.6-8.5, 1.8-8, 2.0-7.5, 2.2-7,2.4-6.5, 2.6-6.0, 2.8-5.5, 3.0-5.0, 3.2-4.5, 3.4-4, and 2-4 μg/L copper.In yet other embodiments, the basal cell culture media used in methodsof the present invention are supplemented to result in about 1-6, 2-5,3-4 μg/L copper. In one embodiment, the basal cell culture media issupplemented with copper to result in a total copper concentration of atleast 1 μg/L. In another embodiment, the basal cell culture media issupplemented with copper to result in a total copper concentration of atleast 2 μg/L. In yet another embodiment, the basal cell culture media issupplemented with copper to result in a total copper concentration of atleast 4 μg/L. In certain embodiments, the basal cell culture media issupplemented with copper to result in a total copper concentration of atleast 1 μg/L, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 μg/L copper, or more. In certain embodiments andas discussed in further detail herein, cultures for producing rA13 maycontain from about 2-4 μg/L of copper, whereas cultures for producingrVWF may contain at least 2 μg/L of copper.

The concentrations indicated above are the respective concentrations ofpure copper, in cupric form (Cu²⁺). If a copper derivative, e.g., ahydrated salt, or a compound comprising copper, e.g., a copper chelator,is used, the amount of derivative or chelator is added such that thefinal concentration of the copper is in the ranges described herein. Forexample, 2 μg/L of CuSO₄.5H₂O is equivalent to a copper concentration ofabout 0.51 μg/L (without sulfate and 5H₂O).

Advantageously, it has been found that the use of cell culture medium incell culture processes resulting in low ammonium (NH₄ ⁺) concentrationsin the cell culture solution (i.e., in the culture supernatant) resultsin the expression of recombinant VWF and/or rA13 with higher specificactivities. Accordingly, in certain embodiments, the NH₄ ⁺ concentrationof the supernatant is no higher than 10 mM. In a preferred embodiment,the NH₄ ⁺ concentration of the supernatant is no higher than 5 mM. In apreferred embodiment, the NH₄ ⁺ concentration of the supernatant is nohigher than 4 mM. In yet other embodiments, the NH₄ ⁺ concentration ofthe supernatant is no higher than 10 mM, 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4mM, 3 mM, 2 mM, 1 mM, or less.

Accordingly, in certain embodiments, the methods and compositionsprovided herein rely on the use of basal cell culture media supplementedwith copper (e.g., to a final concentration of at least 2 μg/L) used ina process that results in an NH₄ ⁺ concentration of no higher than 10 mMin the supernatant. In yet other embodiments, the basal cell culturemedium is supplemented to provide a final copper and ammoniumconcentration according to any one of variations 1 to 440, as providedin Table 1.

TABLE 1 Exemplary embodiments of copper and ammonium concentrationspresent in culture media and supernatant useful for the expression of arecombinant proteins as provided herein. Ammonium Concentration NMT NMTNMT NMT NMT NMT NMT NMT NMT NMT N.D. 10 mM 9 mM 8 mM 7 mM 6 mM 5 mM 4 mM3 mM 2 mM 1 mM Copper AL Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. Concentration 1 μg/L 1 41 81 121 161 201 241 281 321 361 401AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 2 μg/L 2 42 82122 162 202 242 282 322 362 402 AL Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 3 μg/L 3 43 83 123 163 203 243 283 323 363 403 ALVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 4 μg/L 4 44 84124 164 204 244 284 324 364 404 AL Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 5 μg/L 5 45 85 125 165 205 245 285 325 365 405 ALVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 6 μg/L 6 46 86126 166 206 246 286 326 366 406 AL Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 7 μg/L 7 47 87 127 167 207 247 287 327 367 407 ALVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 8 μg/L 8 48 88128 168 208 248 288 328 368 408 AL Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 9 μg/L 9 49 89 129 169 209 249 289 329 369 409 ALVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 10 μg/L 10 50 90130 170 210 250 290 330 370 410 About Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 1 μg/L 11 51 91 131 171 211 251 291 331 371 411About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 1.5 μg/L 1252 92 132 172 212 252 292 332 372 412 About Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 2 μg/L 13 53 93 133 173 213 253 293 333373 413 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 2.5μg/L 14 54 94 134 174 214 254 294 334 374 414 About Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 3 μg/L 15 55 95 135 175 215 255 295335 375 415 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.3.5 μg/L 16 56 96 136 176 216 256 296 336 376 416 About Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 4 μg/L 17 57 97 137 177 217 257297 337 377 417 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 4.5 μg/L 18 58 98 138 178 218 258 298 338 378 418 About Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 5 μg/L 19 59 99 139 179 219259 299 339 379 419 About Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. 5.5 μg/L 20 60 100 140 180 220 260 300 340 380 420 About Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 6 μg/L 21 61 101 141181 221 261 301 341 381 421 About Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 7 μg/L 22 62 102 142 182 222 262 302 342 382 422About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 8 μg/L 2363 103 143 183 223 263 303 343 383 423 About Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 9 μg/L 24 64 104 144 184 224 264 304 344384 424 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 10μg/L 25 65 105 145 185 225 265 305 345 385 425 1-20 μg/L Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 26 66 106 146 186 226 266 306346 386 426 2-20 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 27 67 107 147 187 227 267 307 347 387 427 1-10 μg/L Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 28 68 108 148 188 228 268 308348 388 428 2-10 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 29 69 109 149 189 229 269 309 349 389 429 1-6 μg/L Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 30 70 110 150 190 230 270 310350 390 430 2-6 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 31 71 111 151 191 231 271 311 351 391 431 3-6 μg/L Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 32 72 112 152 192 232 272 312352 392 432 4-6 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 33 73 113 153 193 233 273 313 353 393 433 1-5 μg/L Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 34 74 114 154 194 234 274 314354 394 434 2-5 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 35 75 115 155 195 235 275 315 355 395 435 3-5 μg/L Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 36 76 116 156 196 236 276 316356 396 436 4-5 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 37 77 117 157 197 237 277 317 357 397 437 1-4 μg/L Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 38 78 118 158 198 238 278 318358 398 438 2-4 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 39 79 119 159 199 239 279 319 359 399 439 3-4 μg/L Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 40 80 120 160 200 240 280 320360 400 440 *ND = not defined *NMT = no more than *AL = at least

In some embodiments, the copper supplemented media of the presentinvention are produced by supplementing basal media that is animalprotein-free and/or chemically defined. Methods of preparing animalprotein-free and chemically defined culture mediums are known in theart, for example in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO2007/077217, and U.S. Patent Application Publication Numbers2008/0009040 and 2007/0212770, the disclosures of which are incorporatedherein by reference in their entireties for all purposes. In oneembodiment, the basal culture medium used in the methods describedherein is animal protein-free or oligopeptide-free medium. In certainembodiments, the culture medium may be chemically defined. In certainembodiments, the culture media may contain at least one polyamine at aconcentration of about 0.5 mg/L to about 10 mg/L.

In further embodiments and in addition to any of the descriptionprovided above, culture media of the invention are provided in which abasal medium is supplemented with copper and at least one of calcium,zinc, and/or vitamin B3. In certain embodiments, the medium may be ananimal protein-free, oligopeptide-free, or chemically defined medium. Incertain embodiments, the animal protein-free or oligopeptide free mediumis prepared as taught in U.S. Pat. Nos. 6,171,825 and 6,936,441, WO2007/077217, and U.S. Patent Application Publication Numbers2008/0009040 and 2007/0212770, the disclosures of which are incorporatedherein by reference in their entireties for all purposes, both of whichare incorporated herein by reference in their entireties for allpurposes, and supplemented with additional copper and optionally one ormore of calcium, zinc, and vitamin B3. In a specific embodiment, thechemically defined culture medium may be similar to a Dulbecco'sModified Eagle's Media/Ham's F12 1:1 mixture (DMEM/Ham's F12), which hasbeen supplemented with additional copper and optionally calcium, zinc,and/or vitamin B3, in order to increase the specific activity of rVWF orrA13 expressed in a cell cultured in the medium. In yet otherembodiments, the culture medium is animal component free. In anotherembodiment, the culture medium contains protein, e.g., animal proteinfrom serum such as fetal calf serum. In another embodiment, the culturehas recombinant proteins exogenously added. In another embodiment, theproteins are from a certified pathogen free animal.

In certain embodiments, the culture media contains at least onepolyamine at a concentration of at or about between 0.5 mg/L and 30mg/L. In another embodiment, the culture medium contains at least onepolyamine at or about between 0.5 mg/L and 10 mg/L. In one embodiment,the culture medium contains at least one polyamine at or about between 2mg/L and 8 mg/L. In certain embodiments the polyamine is from the groupof ornithine, putrescine, spermine or spermidine, or the like. In apreferred embodiment, the polyamine is putrescine. In a specificembodiment, the culture medium contains at or about between 2 mg/L and 8mg/L putrescine.

In one embodiment, the culture media contains at least one polyamine ata concentration of at or about between 0.5 mg/L and 30 mg/L and a copperand ammonium combination according to any one of variations 1 to 440, asset forth in Table 1. In another embodiment, the culture medium containsat least one polyamine at or about between 0.5 mg/L and 10 mg/L and acopper and ammonium combination according to any one of variations 1 to440, as set forth in Table 1. In one embodiment, the culture mediumcontains at least one polyamine at or about between 2 mg/L and 8 mg/Land a copper and ammonium combination according to any one of variations1 to 440, as set forth in Table 1. In certain embodiments the polyamineis from the group of ornithine, putrescine, spermine or spermidine, orthe like. In a preferred embodiment, the polyamine is putrescine. In aspecific embodiment, the culture medium contains at or about between 2mg/L and 8 mg/L putrescine and a copper and ammonium combinationaccording to any one of variations 1 to 440, as set forth in Table 1.

In further aspects, in addition to copper, cell culture media of use inthe present invention may further include one or more of: additionalcalcium, zinc, one or more vitamins, and any combination thereof.

Generally, any calcium salt may be used to supplement the media of theinvention, non-limiting examples of acceptable salts include CaCl₂,CaCl₂, CaFPO₃.2H₂O, CaI₂, CaBr₂, (C₂H₃O₂)₂Ca, (CHO₂)₂Ca, (C₆H₇O₆)₂Ca,(C₆H₅O₇)₂Ca₃.2H₂O, and the like. In certain embodiments, apharmaceutically acceptable salt of calcium is used to supplement theculture mediums of the invention.

Generally, any zinc salt may be used to supplement the media of theinvention, non-limiting examples of acceptable salts include,ZnSO₄.7H₂O, ZnSO₃.2H₂O, (C₆H₅O₇)₂Zn₃.2H₂O, ZnBr₂, ZnBr₂.2H₂O, ZnCl₂, Zn(NO₃)₂.6H₂O, Zn(H₂PO₄)₂.H₂O, (C₂H₃O₂)₂Zn₃.2H₂O, and the like. In certainembodiments, a pharmaceutically acceptable salt of zinc is used tosupplement the culture mediums of the invention. In other embodiments, azinc containing peptide or protein preparation, for example insulin, maybe used to the supplement the culture provided herein.

In still further aspects, basal cell media supplemented with copper andone or more of the additional materials discussed above may further beused in cultures with low ammonium levels in the supernatant. In certainembodiments, supplemented cell culture media of use in the presentinvention result in ammonium levels in the cell culture solution ofunder 10 mM. In further embodiments, the supplemented cell culture mediaof the present invention is used with cell culture ammonium levels fromabout 0.5-9.5, 1.0-9.0, 1.5-8.5, 2.0-8.0, 2.5-7.5, 3.0-7.0, 3.5-6.5,4.0-6.0, 4.5-5.5 mM.

In one embodiment, the copper and ammonium concentrations of a cellculture media and a cell culture supernatant are maintained for anextended period of time during the manufacturing process. In a specificembodiment, the copper and ammonium concentrations of a cell culture aremaintained for the duration of a manufacturing process, i.e., during thetime in which rVWF or rA13 is being expressed and recovered from alarge-scale cell culture. In certain embodiments, the copper andammonium concentrations are maintained in the culture solution at alevel according to any one of variations 1 to 440, as set forth inTable 1. In a preferred embodiment the copper and ammoniumconcentrations are maintained for the entire time of such a productionprocess.

In some embodiments, the culture medium provided by the invention may beprovided in a liquid or a dry or powder form. The medium may bepre-aliquoted in an amount suitable for single use or provided in alarger quantity that may be used for more than one cell-culture.Generally, the medium of the invention will be provided in a sterilefashion.

Specific details of cell culture media of use for producing rVWF or rA13are discussed below. Although the following are discussed with respectto either rVWF or rA13, it will be appreciated that any of thediscussion provided below with respect to rVWF is applicable for rA13,and vice versa.

A. Recombinant VWF Cell Culture Media

One aspect of the present invention relates to a cell culture solutionfor producing recombinant vWF, more specifically, high molecular weightvWF having a high specific activity, which are further described herein.In one embodiment, the present invention provides a cell culturesolution for producing high molecular weight, recombinant vWF,comprising a cell culture medium comprising a copper concentration of atleast about 2.4 μg/L and a plurality of cells expressing highlymultimeric vWF comprising about 14 to about 22 dimers and a specificRistocetin co-factor activity of at least about 30 mU/μg.

In one embodiment, the cell culture solution further comprises anammonium concentration of less than 10 mM. In a preferred embodiment,the cell culture comprises an ammonium concentration of no more than 5mM. In yet other embodiments, the cell culture comprises an ammoniumconcentration of no more than 10 mM, or no more than 9 mM, 8 mM, 7 mM, 6mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less. In yet other embodiments, thecell culture has a copper and ammonium concentration according to anyone of variations 1 to 440, as set forth in Table 1. In certainembodiments, the ammonium concentration of the cell culture ismaintained for an extended period at a concentration as provided above.For example, in one embodiment, the ammonium concentration is maintainedat a low concentration for at least 3 days, or at least 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 28, 35, 42, 49, 56,63, 70, or more days. In a specific embodiment, the ammoniumconcentration of the cell culture is maintained at an ammoniumconcentration of no more than 5 mM for at least 7 days. In anotherspecific embodiment, the ammonium concentration of the cell culture ismaintained at an ammonium concentration of no more than 4 mM for atleast 7 days. In a specific embodiment, the ammonium concentration ofthe cell culture is maintained at an ammonium concentration of no morethan 5 mM for at least 14 days. In another specific embodiment, theammonium concentration of the cell culture is maintained at an ammoniumconcentration of no more than 4 mM for at least 14 days. In yet anotherembodiment, the ammonium concentration of the cell culture is maintainedat a low level for the duration of the process (i.e., for the entiretime the culture is being used to produce rVWF).

In one embodiment of the invention, the cell culture media can comprisea copper concentration of at least about 2.4 μg/L, in another embodimentat least about 3 μg/L, in yet another embodiment at least about 4 μg/L,in yet another embodiment at least about 8 μg/L, in yet anotherembodiment at least about 10 μg/L, in yet another embodiment at leastabout 15 μg/L, and in a further embodiment at least about 20 μg/L.

In other embodiments, the copper concentration in the cell culture mediaof the present invention can range from about 2.4 μg/L to about 20 μg/L,in another embodiment from about 2.4 μg/L to about 15 μg/L, in yetanother embodiment from about 2.4 μg/L to about 10 μg/L, in yet anotherembodiment from about 2.4 μg/L to about 8 μg/L, in yet anotherembodiment from about 2.4 μg/L to about 6 μg/L, in yet anotherembodiment from about 2.4 μg/L to about 4 μg/L, in yet anotherembodiment from about 4 μg/L to about 20 μg/L, in yet another embodimentfrom about 4 μg/L to about 15 μg/L, in yet another embodiment from about4 μg/L to about 10 μg/L, in yet another embodiment from about 4 μg/L toabout 8 μg/L, and in a further embodiment from about 4 μg/L to about 6μg/L.

The present invention also provides kits for the expression orproduction of rVWF, the kits comprising a culture medium suitable forthe expression of rVWF having high specific activity.

B. ADAMTS13 (A13) Cell Culture Media

In one aspect, the present invention provides culture media that areuseful for the expression of ADAMTS proteins having high specificactivities. Advantageously, it has been found that by supplementing aculture medium with copper, that the activities of recombinant ADAMTS(e.g., rADAMTS13) enzymes expressed in cells cultured in thesupplemented medium are greatly enhanced, while the enzymes areexpressed at levels as high, if not higher, than cells cultured innon-supplemented mediums.

In one aspect, the present invention provides cell culture mediasupplemented with copper for the expression of recombinant ADAMTS13protein with high specific activity. In one embodiment, the media aresupplemented to result in a total copper concentration of from about 2to about 4 μg/L. In further embodiments, the media are supplemented toresults in a total copper concentration of from about 1-3, 2-3, 3-4μg/L. In one embodiment, the media contain a copper concentration of atleast 1 μg/L. In another embodiment, the media contains at least 2 μg/Lcopper. In another embodiment, the media contains at least 4 μg/Lcopper. In other embodiments, the media contains between 2 μg/L and 20μg/L copper. In another embodiment, the media contains between 1 μg/Land 6 μg/L copper. In another embodiment, the media contains between 2μg/L and 5 μg/L copper. In another embodiment, the media containsbetween 3 μg/L and 4 μg/L copper. In yet other embodiments, the mediacontains at least 1 μg/L copper, or at least 2 μg/L, 3 μg/L, 4 μg/L, 5μg/L, 6 μg/L, 7 μg/L, 8 μg/L, 9 μg/L, 10 μg/L, 11 μg/L, 12 μg/L, 13μg/L, 14 μg/L, 15 μg/L, 16 μg/L, 17 μg/L, 18 μg/L, 19 μg/L, 20 μg/L, orhigher concentrations of copper.

In one embodiment, the cell culture solution further comprises anammonium concentration of less than 10 mM. In a preferred embodiment,the cell culture solution comprises an ammonium concentration of no morethan 5 mM. In yet other embodiments, the cell culture solution comprisesan ammonium concentration of no more than 10 mM, or no more than 9 mM, 8mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less. In yet otherembodiments, the cell culture solution has a copper and ammoniumconcentration according to any one of variations 1 to 440, as set forthin Table 1. In certain embodiments, the ammonium concentration of thecell culture is maintained for an extended period at a concentration asprovided above. For example, in one embodiment, the ammoniumconcentration is maintained at a low concentration for at least 3 days,or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 28, 35, 42, 49, 56, 63, 70, or more days. In a specificembodiment, the ammonium concentration of the cell culture is maintainedat an ammonium concentration of no more than 5 mM for at least 7 days.In another specific embodiment, the ammonium concentration of the cellculture is maintained at an ammonium concentration of no more than 4 mMfor at least 7 days. In a specific embodiment, the ammoniumconcentration of the cell culture is maintained at an ammoniumconcentration of no more than 5 mM for at least 14 days. In anotherspecific embodiment, the ammonium concentration of the cell culture ismaintained at an ammonium concentration of no more than 4 mM for atleast 14 days. In yet another embodiment, the ammonium concentration ofthe cell culture is maintained at a low level for the duration of theprocess (i.e., for the entire time the culture is being used to producerA13).

In one embodiment, a culture medium is provided for the expression of arecombinant ADAMTS protein (e.g., rADAMTS13) containing at least 1 μg/Lcopper and at least 2 μM zinc. In other embodiments, the media containsat least 2 μg/L copper or at least 4 μg/L copper. In another embodimentwherein the media is supplemented with copper, the culture medium alsocontains at least at or about 5 μM zinc. In one embodiment, the culturemedium also contains at or about between 2 μM and 12 μM zinc. In anotherembodiment, the culture medium also contains at or about between 5 μMand 12 μM zinc. In yet other embodiments, the culture medium also maycontain at least at or about 2 μM, or at least at or about 3 μM, 4 μM, 5μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20μM, 25 μM, 30 μM, or more zinc. In one embodiment, the culture mediumcontains a copper and zinc concentration according to any one ofvariations 441 to 880, as set forth in Table 2.

TABLE 2 Exemplary embodiments of copper and zinc concentrations presentin culture media useful for the expression of a recombinant ADAMTS13protein. Zinc Concentration AL AL AL AL AL AL AL AL AL 2-12 5-12 2 μM 3μM 4 μM 5 μM 6 μM 7 μM 8 μM 9 μM 10 μM μM μM Copper AL Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. Concentration 1 μg/L 441 481 521561 601 641 681 721 761 801 841 AL Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 2 μg/L 442 482 522 562 602 642 682 722 762 802 842AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 3 μg/L 443 483523 563 603 643 683 723 763 803 843 AL Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 4 μg/L 444 484 524 564 604 644 684 724 764 804844 AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 5 μg/L 445485 525 565 605 645 685 725 765 805 845 AL Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 6 μg/L 446 486 526 566 606 646 686 726 766 806846 AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 7 μg/L 447487 527 567 607 647 687 727 767 807 847 AL Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 8 μg/L 448 488 528 568 608 648 688 728 768 808848 AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 9 μg/L 449489 529 569 609 649 689 729 769 809 849 AL Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 10 μg/L 450 490 530 570 610 650 690 730 770 810850 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 1 μg/L451 491 531 571 611 651 691 731 771 811 851 About Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 1.5 μg/L 452 492 532 572 612 652 692732 772 812 852 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 2 μg/L 453 493 533 573 613 653 693 733 773 813 853 About Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 2.5 μg/L 454 494 534 574614 654 694 734 774 814 854 About Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 3 μg/L 455 495 535 575 615 655 695 735 775 815 855About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 3.5 μg/L456 496 536 576 616 656 696 736 776 816 856 About Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 4 μg/L 457 497 537 577 617 657 697737 777 817 857 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 4.5 μg/L 458 498 538 578 618 658 698 738 778 818 858 About Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 5 μg/L 459 499 539 579619 659 699 739 779 819 859 About Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 5.5 μg/L 460 500 540 580 620 660 700 740 780 820 860About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 6 μg/L 461501 541 581 621 661 701 741 781 821 861 About Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 7 μg/L 462 502 542 582 622 662 702 742 782822 862 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 8μg/L 463 503 543 583 623 663 703 743 783 823 863 About Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 9 μg/L 464 504 544 584 624 664704 744 784 824 864 About Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. 10 μg/L 465 505 545 585 625 665 705 745 785 825 865 1-20 μg/LVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 466 506 546 586626 666 706 746 786 826 866 2-20 μg/L Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 467 507 547 587 627 667 707 747 787 827 867 1-10μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 468 508 548588 628 668 708 748 788 828 868 2-10 μg/L Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 469 509 549 589 629 669 709 749 789 829 869 1-6μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 470 510 550590 630 670 710 750 790 830 870 2-6 μg/L Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 471 511 551 591 631 671 711 751 791 831 871 3-6μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 472 512 552592 632 672 712 752 792 832 872 4-6 μg/L Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 473 513 553 593 633 673 713 753 793 833 873 1-5μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 474 514 554594 634 674 714 754 794 834 874 2-5 μg/L Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 475 515 555 595 635 675 715 755 795 835 875 3-5μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 476 516 556596 636 676 716 756 796 836 876 4-5 μg/L Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 477 517 557 597 637 677 717 757 797 837 877 1-4μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 478 518 558598 638 678 718 758 798 838 878 2-4 μg/L Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 479 519 559 599 639 679 719 759 799 839 879 3-4μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 480 520 560600 640 680 720 760 800 840 880 *AL = at least

In one embodiment, the cell culture solution further comprises a lowammonium concentration. In one embodiment, the cell culture solutioncomprises an ammonium concentration of less than 10 mM and a copper andzinc concentration according to any one of variations 441 to 880, as setforth in Table 2. In a specific embodiment, the ammonium is maintainedat a concentration of no more than 10 mM for at least 7 days. In apreferred embodiment, the cell culture solution comprises an ammoniumconcentration of no more than 6 mM and a copper and zinc concentrationaccording to any one of variations 441 to 880, as set forth in Table 2.In a specific embodiment, the ammonium is maintained at a concentrationof no more than 6 mM for at least 7 days. In another preferredembodiment, the cell culture solution comprises an ammoniumconcentration of no more than 5 mM and a copper and zinc concentrationaccording to any one of variations 441 to 880, as set forth in Table 2.In a specific embodiment, the ammonium is maintained at a concentrationof no more than 5 mM for at least 7 days. In another preferredembodiment, the cell culture solution comprises an ammoniumconcentration of no more than 4 mM and a copper and zinc concentrationaccording to any one of variations 441 to 880, as set forth in Table 2.In a specific embodiment, the ammonium is maintained at a concentrationof no more than 4 mM for at least 7 days. In yet other embodiments, thecell culture solution comprises an ammonium concentration of no morethan 10 mM, or no more than 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2mM, 1 mM, or less and a copper and zinc concentration according to anyone of variations 441 to 880, as set forth in Table 2. In yet anotherspecific embodiment, the ammonium concentration of the cell culture ismaintained at a low level for the duration of the process (i.e., for theentire time the culture is being used to produce rA13).

In one embodiment, a culture medium is provided for the expression of arecombinant ADAMTS protein (e.g., rADAMTS13) containing at least 1 μg/Lcopper and at least at or about 0.5 mM calcium. In other embodiments,the media contains at least 2 μg/L copper or at least 4 μg/L copper. Inanother embodiment wherein the media is supplemented with copper, theculture medium also contains at least 1.5 mM calcium. In one embodiment,the culture medium contains at or about between 0.5 mM and 1.5 mMcalcium. In yet other embodiments, the culture medium may contain atleast at or about 0.5 mM, or at least at or about 0.6 mM, 0.7 mM, 0.8mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM,4.0 mM, 4.5 mM, 5.0 mM, or more calcium. In one embodiment, the culturemedium contains a copper and calcium concentration according to any oneof variations 881 to 1320, as set forth in Table 3.

TABLE 3 Exemplary embodiments of copper and calcium concentrationspresent in culture media useful for the expression of a recombinantADAMTS13 protein. Calcium Concentration AL AL AL AL AL AL AL AL AL AL0.5 0.75 1.0 1.25 1.5 2.0 2.5 3.0 4 5 0.5-1.5 mM mM mM mM mM mM mM mM mMmM mM Copper AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Concentration 1 μg/L 881 921  961 1001 1041 1081 1121 1161 1201 12411281 AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 2 μg/L882 922  962 1002 1042 1082 1122 1162 1202 1242 1282 AL Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 3 μg/L 883 923  963 1003 10431083 1123 1163 1203 1243 1283 AL Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. 4 μg/L 884 924  964 1004 1044 1084 1124 1164 1204 12441284 AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 5 μg/L885 925  965 1005 1045 1085 1125 1165 1205 1245 1285 AL Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 6 μg/L 886 926  966 1006 10461086 1126 1166 1206 1246 1286 AL Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. 7 μg/L 887 927  967 1007 1047 1087 1127 1167 1207 12471287 AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 8 μg/L888 928  968 1008 1048 1088 1128 1168 1208 1248 1288 AL Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 9 μg/L 889 929  969 1009 10491089 1129 1169 1209 1249 1289 AL Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. 10 μg/L 890 930  970 1010 1050 1090 1130 1170 1210 12501290 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 1 μg/L891 931  971 1011 1051 1091 1131 1171 1211 1251 1291 About Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 1.5 μg/L 892 932  972 10121052 1092 1132 1172 1212 1252 1292 About Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 2 μg/L 893 933  973 1013 1053 1093 1133 11731213 1253 1293 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 2.5 μg/L 894 934  974 1014 1054 1094 1134 1174 1214 1254 1294 AboutVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 3 μg/L 895 935 975 1015 1055 1095 1135 1175 1215 1255 1295 About Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 3.5 μg/L 896 936  976 1016 1056 10961136 1176 1216 1256 1296 About Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. 4 μg/L 897 937  977 1017 1057 1097 1137 1177 1217 12571297 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 4.5μg/L 898 938  978 1018 1058 1098 1138 1178 1218 1258 1298 About Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 5 μg/L 899 939  9791019 1059 1099 1139 1179 1219 1259 1299 About Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 5.5 μg/L 900 940  980 1020 1060 1100 11401180 1220 1260 1300 About Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. 6 μg/L 901 941  981 1021 1061 1101 1141 1181 1221 1261 1301About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 7 μg/L 902942  982 1022 1062 1102 1142 1182 1222 1262 1302 About Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 8 μg/L 903 943  983 1023 10631103 1143 1183 1223 1263 1303 About Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 9 μg/L 904 944  984 1024 1064 1104 1144 1184 12241264 1304 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.10 μg/L 905 945  985 1025 1065 1105 1145 1185 1225 1265 1305 1-20 μg/LVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 906 946  986 10261066 1106 1146 1186 1226 1266 1306 2-20 μg/L Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 907 947  987 1027 1067 1107 1147 1187 12271267 1307 1-10 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 908 948  988 1028 1068 1108 1148 1188 1228 1268 1308 2-10 μg/L Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 909 949  989 1029 10691109 1149 1189 1229 1269 1309 1-6 μg/L Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 910 950  990 1030 1070 1110 1150 1190 1230 12701310 2-6 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 911951  991 1031 1071 1111 1151 1191 1231 1271 1311 3-6 μg/L Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 912 952  992 1032 1072 1112 11521192 1232 1272 1312 4-6 μg/L Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. 913 953  993 1033 1073 1113 1153 1193 1233 1273 1313 1-5μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 914 954  9941034 1074 1114 1154 1194 1234 1274 1314 2-5 μg/L Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 915 955  995 1035 1075 1115 1155 11951235 1275 1315 3-5 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. 916 956  996 1036 1076 1116 1156 1196 1236 1276 1316 4-5 μg/LVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 917 957  997 10371077 1117 1157 1197 1237 1277 1317 1-4 μg/L Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 918 958  998 1038 1078 1118 1158 1198 12381278 1318 2-4 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 919 959  999 1039 1079 1119 1159 1199 1239 1279 1319 3-4 μg/L Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 920 960 1000 1040 10801120 1160 1200 1240 1280 1320 *AL = at least

In one embodiment, the cell culture solution further comprises a lowammonium concentration. In one embodiment, the cell culture solutioncomprises an ammonium concentration of less than 10 mM and a copper andcalcium concentration according to any one of variations 881 to 1320, asset forth in Table 3. In a specific embodiment, the ammonium ismaintained at a concentration of no more than 10 mM for at least 7 days.In a preferred embodiment, the cell culture medium comprises an ammoniumconcentration of no more than 6 mM and a copper and calciumconcentration according to any one of variations 881 to 1320, as setforth in Table 3. In a specific embodiment, the ammonium is maintainedat a concentration of no more than 6 mM for at least 7 days. In anotherpreferred embodiment, the cell culture medium comprises an ammoniumconcentration of no more than 5 mM and a copper and calciumconcentration according to any one of variations 881 to 1320, as setforth in Table 3. In a specific embodiment, the ammonium is maintainedat a concentration of no more than 5 mM for at least 7 days. In anotherpreferred embodiment, the cell culture medium comprises an ammoniumconcentration of no more than 4 mM and a copper and calciumconcentration according to any one of variations 881 to 1320, as setforth in Table 3. In a specific embodiment, the ammonium is maintainedat a concentration of no more than 4 mM for at least 7 days. In yetother embodiments, the cell culture medium comprises an ammoniumconcentration of no more than 10 mM, or no more than 9 mM, 8 mM, 7 mM, 6mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less and a copper and calciumconcentration according to any one of variations 881 to 1320, as setforth in Table 3. In yet another specific embodiment, the ammoniumconcentration of the cell culture is maintained at a low level for theduration of the process (i.e., for the entire time the culture is beingused to produce rA13).

In one embodiment, the cell culture medium is supplemented with copper,zinc and calcium. In a specific embodiment, the culture medium has acalcium concentration of at least 0.5 mM and a copper and zincconcentration according to any one of variations 441 to 880, as setforth in Table 2. In another specific embodiment, the culture medium hasa calcium concentration of at least 1.5 mM and a copper and zincconcentration according to any one of variations 441 to 880, as setforth in Table 2. In another specific embodiment, the culture medium hasa calcium concentration between 0.5 mM and 1.5 mM and a copper and zincconcentration according to any one of variations 441 to 880, as setforth in Table 2. In yet other embodiments, the culture medium has acalcium concentration of at least 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM,5.0 mM, or more, and a copper and zinc concentration according to anyone of variations 441 to 880, as set forth in Table 2.

In one embodiment, a culture medium is provided for the expression of arecombinant ADAMTS protein (e.g., rADAMTS13) containing at least 1 μg/Lcopper and at least 2 mg/L nicotinamide (vitamin B3). In otherembodiments, the media contains at least 2 μg/L copper or at least 4μg/L copper. In another embodiment wherein the media is supplementedwith copper, the culture medium also contains at least 7 mg/Lnicotinamide (vitamin B3). In one embodiment, the culture mediumcontains at or about between 2 mg/L and 10 mg/L nicotinamide (vitaminB3). In yet other embodiments, the culture medium may contain at leastat or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8 mg/L, 9mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higher concentrations ofnicotinamide (vitamin B3). In one embodiment, the culture mediumcontains a copper and nicotinamide concentration according to any one ofvariations 1321 to 1760, as set forth in Table 4.

TABLE 4 Exemplary embodiments of copper and nicotinamide concentrationspresent in culture media useful for the expression of a recombinantADAMTS13 protein. Calcium Concentration AL AL AL AL AL AL AL AL AL AL 23 4 5 6 7 8 9 10 15 2-10 mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mL mg/mLmg/mL mg/mL mg/mL Copper AL Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. Concentration 1 μg/L 1321 1361 1401 1441 1481 1521 1561 16011641 1681 1721 AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.2 μg/L 1322 1362 1402 1442 1482 1522 1562 1602 1642 1682 1722 AL Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 3 μg/L 1323 1363 14031443 1483 1523 1563 1603 1643 1683 1723 AL Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 4 μg/L 1324 1364 1404 1444 1484 1524 1564 16041644 1684 1724 AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.5 μg/L 1325 1365 1405 1445 1485 1525 1565 1605 1645 1685 1725 AL Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 6 μg/L 1326 1366 14061446 1486 1526 1566 1606 1646 1686 1726 AL Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 7 μg/L 1327 1367 1407 1447 1487 1527 1567 16071647 1687 1727 AL Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.8 μg/L 1328 1368 1408 1448 1488 1528 1568 1608 1648 1688 1728 AL Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 9 μg/L 1329 1369 14091449 1489 1529 1569 1609 1649 1689 1729 AL Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 10 μg/L 1330 1370 1410 1450 1490 1530 1570 16101650 1690 1730 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 1 μg/L 1331 1371 1411 1451 1491 1531 1571 1611 1651 1691 1731 AboutVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 1.5 μg/L 13321372 1412 1452 1492 1532 1572 1612 1652 1692 1732 About Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 2 μg/L 1333 1373 1413 1453 14931533 1573 1613 1653 1693 1733 About Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 2.5 μg/L 1334 1374 1414 1454 1494 1534 1574 16141654 1694 1734 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 3 μg/L 1335 1375 1415 1455 1495 1535 1575 1615 1655 1695 1735 AboutVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 3.5 μg/L 13361376 1416 1456 1496 1536 1576 1616 1656 1696 1736 About Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 4 μg/L 1337 1377 1417 1457 14971537 1577 1617 1657 1697 1737 About Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 4.5 μg/L 1338 1378 1418 1458 1498 1538 1578 16181658 1698 1738 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 5 μg/L 1339 1379 1419 1459 1499 1539 1579 1619 1659 1699 1739 AboutVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 5.5 μg/L 13401380 1420 1460 1500 1540 1580 1620 1660 1700 1740 About Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. 6 μg/L 1341 1381 1421 1461 15011541 1581 1621 1661 1701 1741 About Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. 7 μg/L 1342 1382 1422 1462 1502 1542 1582 1622 16621702 1742 About Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 8μg/L 1343 1383 1423 1463 1503 1543 1583 1623 1663 1703 1743 About Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 9 μg/L 1344 1384 14241464 1504 1544 1584 1624 1664 1704 1744 About Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. 10 μg/L 1345 1385 1425 1465 1505 1545 15851625 1665 1705 1745 1-20 μg/L Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. 1346 1386 1426 1466 1506 1546 1586 1626 1666 1706 17462-20 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 13471387 1427 1467 1507 1547 1587 1627 1667 1707 1747 1-10 μg/L Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 1348 1388 1428 1468 15081548 1588 1628 1668 1708 1748 2-10 μg/L Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 1349 1389 1429 1469 1509 1549 1589 1629 16691709 1749 1-6 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 1350 1390 1430 1470 1510 1550 1590 1630 1670 1710 1750 2-6 μg/LVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 1351 1391 14311471 1511 1551 1591 1631 1671 1711 1751 3-6 μg/L Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 1352 1392 1432 1472 1512 1552 15921632 1672 1712 1752 4-6 μg/L Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. 1353 1393 1433 1473 1513 1553 1593 1633 1673 1713 17531-5 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 13541394 1434 1474 1514 1554 1594 1634 1674 1714 1754 2-5 μg/L Var. Var.Var. Var. Var. Var. Var. Var. Var. Var. Var. 1355 1395 1435 1475 15151555 1595 1635 1675 1715 1755 3-5 μg/L Var. Var. Var. Var. Var. Var.Var. Var. Var. Var. Var. 1356 1396 1436 1476 1516 1556 1596 1636 16761716 1756 4-5 μg/L Var. Var. Var. Var. Var. Var. Var. Var. Var. Var.Var. 1357 1397 1437 1477 1517 1557 1597 1637 1677 1717 1757 1-4 μg/LVar. Var. Var. Var. Var. Var. Var. Var. Var. Var. Var. 1358 1398 14381478 1518 1558 1598 1638 1678 1718 1758 2-4 μg/L Var. Var. Var. Var.Var. Var. Var. Var. Var. Var. Var. 1359 1399 1439 1479 1519 1559 15991639 1679 1719 1759 3-4 μg/L Var. Var. Var. Var. Var. Var. Var. Var.Var. Var. Var. 1360 1400 1440 1480 1520 1560 1600 1640 1680 1720 1760*AL = at least

In one embodiment, the cell culture solution further comprises a lowammonium concentration. In one embodiment, the culture medium comprisesan ammonium concentration of less than 10 mM and a copper andnicotinamide concentration according to any one of variations 1321 to1760, as set forth in Table 4. In a specific embodiment, the ammonium ismaintained at a concentration of no more than 10 mM for at least 7 days.In a preferred embodiment, the cell culture solution comprises anammonium concentration of no more than 6 mM and a copper andnicotinamide concentration according to any one of variations 1321 to1760, as set forth in Table 4. In a specific embodiment, the ammonium ismaintained at a concentration of no more than 6 mM for at least 7 days.In another preferred embodiment, the cell culture solution comprises anammonium concentration of no more than 5 mM and a copper andnicotinamide concentration according to any one of variations 1321 to1760, as set forth in Table 4. In a specific embodiment, the ammonium ismaintained at a concentration of no more than 5 mM for at least 7 days.In another preferred embodiment, the cell culture solution comprises anammonium concentration of no more than 4 mM and a copper andnicotinamide concentration according to any one of variations 1321 to1760, as set forth in Table 4. In a specific embodiment, the ammonium ismaintained at a concentration of no more than 4 mM for at least 7 days.In yet other embodiments, the cell culture solution comprises anammonium concentration of no more than 10 mM, or no more than 9 mM, 8mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less and a copper andnicotinamide concentration according to any one of variations 1321 to1760, as set forth in Table 4. In yet another specific embodiment, theammonium concentration of the cell culture is maintained at a low levelfor the duration of the process (i.e., for the entire time the cultureis being used to produce rA13).

In one embodiment, the cell culture medium is supplemented with copper,zinc and nicotinamide. In a specific embodiment, the culture medium hasa nicotinamide concentration of at least 2 mg/mL and a copper and zincconcentration according to any one of variations 441 to 880, as setforth in Table 2. In another specific embodiment, the culture medium hasa nicotinamide concentration of at least 7 mg/mL mM and a copper andzinc concentration according to any one of variations 441 to 880, as setforth in Table 2. In another specific embodiment, the culture medium hasa nicotinamide concentration between 2 mg/mL and 10 mg/mL and a copperand zinc concentration according to any one of variations 441 to 880, asset forth in Table 2. In yet other embodiments, the culture medium has anicotinamide concentration of at least 2 mg/mL, 3 mg/mL, 4 mg/mL, 5mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL,13 mg/mL, 14 mg/mL, 15 mg/mL, or more, and a copper and zincconcentration according to any one of variations 441 to 880, as setforth in Table 2.

In one embodiment, the cell culture medium is supplemented with copper,calcium and nicotinamide. In a specific embodiment, the culture mediumhas a nicotinamide concentration of at least 2 mg/mL and a copper andcalcium concentration according to any one of variations 881 to 1320, asset forth in Table 3. In another specific embodiment, the culture mediumhas a nicotinamide concentration of at least 7 mg/mL mM and a copper andcalcium concentration according to any one of variations 881 to 1320, asset forth in Table 3. In another specific embodiment, the culture mediumhas a nicotinamide concentration between 2 mg/mL and 10 mg/mL and acopper and calcium concentration according to any one of variations 881to 1320, as set forth in Table 3. In yet other embodiments, the culturemedium has a nicotinamide concentration of at least 2 mg/mL, 3 mg/mL, 4mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL,12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, or more, and a copper andcalcium concentration according to any one of variations 881 to 1320, asset forth in Table 3.

IV. METHODS FOR THE PRODUCTION OF BLOOD FACTORS HAVING HIGH SPECIFICACTIVITY

A. Cell Cultivation Methods

The present invention provides methods for large-scale production ofrecombinant proteins (such as rVWF and rA13). In certain embodiments,such large-scale production methods utilize stirred/agitated tankreactors for manufacture of these therapeutic recombinant proteins.

In certain embodiments, the methods of the present invention cancomprise the use of a cell culture system operated under a batch orcontinuous mode of operation. For example, when batch cell cultures areutilized, they may be operated under single batch, fed-batch, orrepeated-batch mode. Likewise, continuous cell cultures may be operatedunder, for example, perfusion, turbidostat or chemostat mode. Batch andcontinuous cell cultivation may be performed under either suspension oradherence conditions. When operated under suspension conditions, thecells will be freely suspended and mixed within the culture medium.Alternatively, under adherence conditions, the cells will be bound to asolid phase, for example, a microcarrier, a porous microcarrier, diskcarrier, ceramic cartridge, hollow fiber, flat sheet, gel matrix, andthe like.

A batch culture is typically a large scale cell culture in which a cellinoculum is cultured to a maximum density in a tank or fermenter, andharvested and processed as a single batch. A fed-batch culture ittypically a batch culture which is supplied with either fresh nutrients(e.g., growth-limiting substrates) or additives (e.g., precursors toproducts). The feed solution is usually highly concentrated to avoiddilution of the bioreactor. In a repeated-batch culture, the cells areplaced in a culture medium and grown to a desired cell density. To avoidthe onset of a decline phase and cell death, the culture is then dilutedwith complete growth medium before the cells reach their maximumconcentration. The amount and frequency of dilution varies widely anddepends on the growth characteristics of the cell line and convenienceof the culture process. The process can be repeated as many times asrequired and, unless cells and medium are discarded at subculture, thevolume of culture will increase stepwise as each dilution is made. Theincreasing volume may be handled by having a reactor of sufficient sizeto allow dilutions within the vessel or by dividing the diluted cultureinto several vessels. The rationale of this type of culture is tomaintain the cells in an exponentially growing state. Serial subcultureis characterized in that the volume of culture is always increasingstepwise, there can be multiple harvests, the cells continue to grow andthe process can continue for as long as desired. In certain embodiments,a recombinant ADAMTS protein (e.g., rADAMTS13) may be recovered afterharvesting the supernatant of a batch culture. In other embodiments, arecombinant VWF may be recovered after harvesting the supernatant of abatch culture.

A continuous culture can be a suspension culture that is continuouslysupplied with nutrients by the inflow of fresh medium, wherein theculture volume is usually kept constant by the concomitant removal ofspent medium. In chemostat and turbidostat methods, the extracted mediumcontains cells. Thus, the cells remaining in the cell culture vesselmust grow to maintain a steady state. In the chemostat method, thegrowth rate is typically controlled by controlling the dilution rate,i.e., the rate at which fresh medium is added. The growth rate of thecells in the culture may be controlled, for example, at a sub-maximalgrowth rate, by alteration of the dilution rate. In contrast, in theturbidostat method, the dilution rate is set to permit the maximumgrowth rate that the cells can achieve at the given operatingconditions, such as pH and temperature. In certain embodiments, the rVWFor rA13 is recovered after harvesting the supernatant of a continuousculture. An exemplary method for continuous cell cultivation isdescribed in WO/2011/012725 (Grillberger et al.), the content of whichis hereby incorporated by reference in its entirety for all purposes.

In a perfusion culture, the extracted medium is depleted of cells, whichare retained in the culture vessel, for example, by filtration or bycentrifugal methods that lead to the reintroduction of the cells intothe culture. However, typically membranes used for filtration do notretain 100% of cells, and so a proportion is removed when the medium isextracted. It may not be crucial to operate perfusion cultures at veryhigh growth rates, as the majority of the cells are retained in theculture vessel. In certain embodiments, the rVWF or rA13 is recoveredafter harvesting the supernatant of a perfusion culture.

Stirred-tank reactor system can be used for batch and continuous cellcultures operated under suspension or adherent modes. Generally, thestirred-tank reactor system can be operated as any conventionalstirred-tank reactor with any type of agitator such as a Rushton,hydrofoil, pitched blade, or marine.

In certain embodiments, the cell-culture methods of the invention maycomprise the use of a microcarrier. In some embodiments, thecell-cultures of the embodiments can be performed in large bioreactorsunder conditions suitable for providing high volume-specific culturesurface areas to achieve high cell densities and protein expression. Onemeans for providing such growth conditions is to use microcarriers forcell-culture in stirred tank bioreactors. The concept of cell-growth onmicrocarriers was first described by van Wezel (van Wezel, A. L., Nature216:64-5 (1967)) and allows for cell attachment on the surface of smallsolid particles suspended in the growth medium. These methods providefor high surface-to-volume ratios and thus allow for efficient nutrientutilization. Furthermore, for expression of secreted proteins ineukaryotic cell lines, the increased surface-to-volume ratio allows forhigher levels of secretion and thus higher protein yields in thesupernatant of the culture. Finally, these methods allow for the easyscale-up of eukaryotic expression cultures.

The cells expressing vWF and/or rA13 can be bound to a spherical or aporous microcarrier during cell culture growth. The microcarrier can bea microcarrier selected from the group of microcarriers based ondextran, collagen, plastic, gelatine and cellulose and others asdescribed in Butler (1988. In: Spier & Griffiths, Animal CellBiotechnology 3:283-303). It is also possible to grow the cells to abiomass on spherical microcarriers and subculture the cells when theyhave reached final fermenter biomass and prior to production of theexpressed protein on a porous microcarrier or vice versa. Suitablespherical microcarriers can include smooth surface microcarriers, suchas Cytodex™ 1, Cytodex™ 2, and Cytodex™ 3 (GE Healthcare) andmacroporous microcarriers such as Cytopore™ 1, Cytopore™ 2, Cytoline™ 1,and Cytoline™ 2 (GE Healthcare).

As described above, the present invention includes cell culture mediahaving an increased copper concentration. It is understood that all ofthe embodiments and concentrations described in the “Cell Culture Media”section above can be applied to the methods of the present inventiondescribed herein.

In certain embodiments, the culture can be maintained for at least about7 days, or at least about 14 days, 21 days, 28 days, or at least about 5weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or at least about 2 months,or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months orlonger. The cell density at which a cell-culture is maintained at forproduction of a recombinant vWF or recombinant rA13 protein will dependupon the culture-conditions and medium used for protein expression. Oneof skill in the art will readily be able to determine the optimal celldensity for a cell-culture producing rVWF or rA13. In one embodiment,the culture is maintained at a cell density of between about 0.5×10⁶ and4×10⁷ cells/mL for an extended period of time. In other embodiments, thecell density is maintained at a concentration of between about 1.0×10⁶and about 1.0×10⁷ cells/mL for an extended period of time. In otherembodiments, the cell density is maintained at a concentration ofbetween about 1.0×10⁶ and about 4.0×10⁶ cells/mL for an extended periodof time. In other embodiments, the cell density is maintained at aconcentration of between about 1.0×10⁶ and about 4.0×10⁶ cells/mL for anextended period of time. In yet other embodiments, the cell density maybe maintained at a concentration between about 2.0×10⁶ and about 4.0×10⁶cells/mL, or between about 1.0×10⁶ and about 2.5×10⁶ cells/mL, orbetween about 1.5×10⁶ and about 3.5×10⁶ cells/mL, or any other similarrange, for an extended period of time.

In one embodiment, the cell density of a continuous cell cultureprovided herein is maintained at a concentration of no more than 4.0×10⁶cells/mL for at least 7 days. In a specific embodiment, the cell densityof a continuous cell culture provided herein is maintained at aconcentration of no more than 4.0×10⁶ cells/mL for at least 14 days. Ina more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than4.0×10⁶ cells/mL for at least 21 days. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 4.0×10⁶ cells/mLfor at least 28 days. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 4.0×10⁶ cells/mL for at least 5 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than4.0×10⁶ cells/mL for at least 6 weeks. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 4.0×10⁶ cells/mLfor at least 7 weeks. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 4.0×10⁶ cells/mL for at least 8 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than4.0×10⁶ cells/mL for at least 9 weeks.

In one embodiment, the cell density of a continuous cell cultureprovided herein is maintained at a concentration of no more than 3.5×10⁶cells/mL for at least 7 days. In a specific embodiment, the cell densityof a continuous cell culture provided herein is maintained at aconcentration of no more than 3.5×10⁶ cells/mL for at least 14 days. Ina more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than3.5×10⁶ cells/mL for at least 21 days. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 3.5×10⁶ cells/mLfor at least 28 days. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 3.5×10⁶ cells/mL for at least 5 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than3.5×10⁶ cells/mL for at least 6 weeks. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 3.5×10⁶ cells/mLfor at least 7 weeks. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 3.5×10⁶ cells/mL for at least 8 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than3.5×10⁶ cells/mL for at least 9 weeks.

In one embodiment, the cell density of a continuous cell cultureprovided herein is maintained at a concentration of no more than 3.0×10⁶cells/mL for at least 7 days. In a specific embodiment, the cell densityof a continuous cell culture provided herein is maintained at aconcentration of no more than 3.0×10⁶ cells/mL for at least 14 days. Ina more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than3.0×10⁶ cells/mL for at least 21 days. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 3.0×10⁶ cells/mLfor at least 28 days. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 3.0×10⁶ cells/mL for at least 5 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than3.0×10⁶ cells/mL for at least 6 weeks. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 3.0×10⁶ cells/mLfor at least 7 weeks. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 3.0×10⁶ cells/mL for at least 8 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than3.0×10⁶ cells/mL for at least 9 weeks.

In one embodiment, the cell density of a continuous cell cultureprovided herein is maintained at a concentration of no more than 2.5×10⁶cells/mL for at least 7 days. In a specific embodiment, the cell densityof a continuous cell culture provided herein is maintained at aconcentration of no more than 2.5×10⁶ cells/mL for at least 14 days. Ina more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than2.5×10⁶ cells/mL for at least 21 days. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 2.5×10⁶ cells/mLfor at least 28 days. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 2.5×10⁶ cells/mL for at least 5 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than2.5×10⁶ cells/mL for at least 6 weeks. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 2.5×10⁶ cells/mLfor at least 7 weeks. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 2.5×10⁶ cells/mL for at least 8 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than2.5×10⁶ cells/mL for at least 9 weeks.

In another embodiment, the cell density of a continuous cell cultureprovided herein is maintained at a concentration of no more than 2.0×10⁶cells/mL for at least 7 days. In a specific embodiment, the cell densityof a continuous cell culture provided herein is maintained at aconcentration of no more than 2.0×10⁶ cells/mL for at least 14 days. Ina more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than2.0×10⁶ cells/mL for at least 21 days. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 2.0×10⁶ cells/mLfor at least 28 days. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 2.0×10⁶ cells/mL for at least 5 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than2.0×10⁶ cells/mL for at least 6 weeks. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 2.0×10⁶ cells/mLfor at least 7 weeks. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 2.0×10⁶ cells/mL for at least 8 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than2.0×10⁶ cells/mL for at least 9 weeks.

In one embodiment, the cell density of a continuous cell cultureprovided herein is maintained at a concentration of no more than 1.5×10⁶cells/mL for at least 7 days. In a specific embodiment, the cell densityof a continuous cell culture provided herein is maintained at aconcentration of no more than 1.5×10⁶ cells/mL for at least 14 days. Ina more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than1.5×10⁶ cells/mL for at least 21 days. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 1.5×10⁶ cells/mLfor at least 28 days. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 1.5×10⁶ cells/mL for at least 5 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than1.5×10⁶ cells/mL for at least 6 weeks. In a yet more specificembodiment, the cell density of a continuous cell culture providedherein is maintained at a concentration of no more than 1.5×10⁶ cells/mLfor at least 7 weeks. In a yet more specific embodiment, the celldensity of a continuous cell culture provided herein is maintained at aconcentration of no more than 1.5×10⁶ cells/mL for at least 8 weeks. Ina yet more specific embodiment, the cell density of a continuous cellculture provided herein is maintained at a concentration of no more than1.5×10⁶ cells/mL for at least 9 weeks.

The following provides specific details on methods for producing rVWFand rA13. As will be appreciated, although the conditions are presentedspecifically for rVWF or rA13, the conditions for rVWF can be used forproducing rA13 and vice versa.

B. Methods of Producing High Molecular Weight Recombinant vWF

In another aspect, the present invention further relates to methods forproducing vWF under cell culture conditions comprising a cell culturemedium having an increased copper concentration. In certain embodiments,the culture also comprises a low ammonium concentration. As used herein,the term “cell culture” and “cell culture solution” are usedinterchangeably.

In one embodiment, the present invention provides a method of producinga high molecular weight, recombinant vWF, comprising: a) providing aculture of cells; b) introducing a nucleic acid sequence coding for vWF;c) selecting the cells carrying the nucleic acid sequence; and, d)expressing vWF in the cells under cell culture conditions comprising acell culture medium comprising a copper concentration of at least about2.4 μg/L and a cell culture supernatant comprising an ammoniumconcentration less than about 10 mM, wherein the vWF is highlymultimeric vWF comprising about 14 to about 22 dimers and a specificRistocetin activity of at least about 30 mU/μg. In further embodiments,the multimeric rVWF produced using methods of the present inventioncomprise about 10-30, 12-28, 14-26, 16-24, 18-22, 20-21 dimers. In stillfurther embodiments, the rVWF produced in accordance with the presentinvention has a specific activity of at least about 20, 22.5, 25, 27.5,30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5,65, 67.5, 70, 72.5, 75, 77.5, 80, or more mU/μg. In a specificembodiment, the cell density of the continuous cell culture forproduction of rVWF is maintained at a concentration of no more than2.5×10⁶ cells/mL for an extended period. In other specific embodiments,the cell density is maintained at no more than 2.0×10⁶ cells/mL, 1.5×10⁶cells/mL, 1.0×10⁶ cells/mL, 0.5×10⁶ cells/mL, or less. In oneembodiment, the cell density is maintained at between 1.5×10⁶ cells/mLand 2.5×10⁶ cells/mL.

In one embodiment, the present invention provides a method for producinga recombinant Von Willebrand Factor (rVWF) composition, the methodcomprising the steps of: (a) providing a basal cell culture media; (b)supplementing the basal cell culture media with copper to provide afinal copper concentration of at least 2.0 μg/L; (c) providing one ormore cells comprising a nucleic acid encoding a rVWF protein; (d)culturing the one or more cells in the copper supplemented cell culturemedia such that rVWF is expressed and excreted from the cells into aculture supernatant; and (e) recovering at least a portion of theculture supernatant, wherein the recovered supernatant has a rVWFspecific ristocetin cofactor activity of at least 30 mU/μg rVWF. In oneembodiment, the cell culture solution further comprises an ammoniumconcentration of less than 10 mM. In another specific embodiment, theammonium concentration of the cell culture solution is maintained at anammonium concentration of no more than 10 mM for at least 7 days. In apreferred embodiment, the cell culture solution comprises an ammoniumconcentration of no more than 5 mM. In another specific embodiment, theammonium concentration of the cell culture solution is maintained at anammonium concentration of no more than 5 mM for at least 7 days. In apreferred embodiment, the cell culture solution comprises an ammoniumconcentration of no more than 4 mM. In another specific embodiment, theammonium concentration of the cell culture is maintained at an ammoniumconcentration of no more than 4 mM for at least 7 days. In yet otherembodiments, the cell culture solution comprises an ammoniumconcentration of no more than 10 mM, or no more than 9 mM, 8 mM, 7 mM, 6mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less. In yet another embodiment,the ammonium concentration of the cell culture is maintained at a lowlevel for the duration of the process (i.e., for the entire time theculture is being used to produce rVWF). In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment of the method described above, the basal cell culturemedia is supplemented with copper to provide a final copperconcentration of at least 2.4 μg/L. In one embodiment, the cell culturesolution further comprises an ammonium concentration of less than 10 mM.In another specific embodiment, the ammonium concentration of the cellculture solution is maintained at an ammonium concentration of no morethan 10 mM for at least 7 days. In a preferred embodiment, the cellculture solution comprises an ammonium concentration of no more than 5mM. In another specific embodiment, the ammonium concentration of thecell culture solution is maintained at an ammonium concentration of nomore than 5 mM for at least 7 days. In a preferred embodiment, the cellculture solution comprises an ammonium concentration of no more than 4mM. In another specific embodiment, the ammonium concentration of thecell culture solution is maintained at an ammonium concentration of nomore than 4 mM for at least 7 days. In yet other embodiments, the cellculture solution comprises an ammonium concentration of no more than 10mM, or no more than 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1mM, or less. In yet another embodiment, the ammonium concentration ofthe cell culture solution is maintained at a low level for the durationof the process (i.e., for the entire time the culture is being used toproduce rVWF). In a preferred embodiment, the recovered supernatant hasa rVWF specific ristocetin cofactor activity of at least 50 mU/μg rVWF.In a more preferred embodiment, the recovered supernatant has a rVWFspecific ristocetin cofactor activity of at least 70 mU/μg rVWF.

In one embodiment of the method described above, the basal cell culturemedia is supplemented with copper to provide a final copperconcentration of at least 3 μg/L. In one embodiment, the cell culturesolution further comprises an ammonium concentration of less than 10 mM.In another specific embodiment, the ammonium concentration of the cellculture solution is maintained at an ammonium concentration of no morethan 10 mM for at least 7 days. In a preferred embodiment, the cellculture solution comprises an ammonium concentration of no more than 5mM. In another specific embodiment, the ammonium concentration of thecell culture solution is maintained at an ammonium concentration of nomore than 5 mM for at least 7 days. In a preferred embodiment, the cellculture solution comprises an ammonium concentration of no more than 4mM. In another specific embodiment, the ammonium concentration of thecell culture solution is maintained at an ammonium concentration of nomore than 4 mM for at least 7 days. In yet other embodiments, the cellculture solution comprises an ammonium concentration of no more than 10mM, or no more than 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1mM, or less. In yet another embodiment, the ammonium concentration ofthe cell culture solution is maintained at a low level for the durationof the process (i.e., for the entire time the culture is being used toproduce rVWF). In a preferred embodiment, the recovered supernatant hasa rVWF specific ristocetin cofactor activity of at least 50 mU/μg rVWF.In a more preferred embodiment, the recovered supernatant has a rVWFspecific ristocetin cofactor activity of at least 70 mU/μg rVWF.

In one embodiment of the method described above, the basal cell culturemedia is supplemented with copper to provide a final copperconcentration of at least 4 μg/L. In one embodiment, the cell culturesolution further comprises an ammonium concentration of less than 10 mM.In another specific embodiment, the ammonium concentration of the cellculture solution is maintained at an ammonium concentration of no morethan 10 mM for at least 7 days. In a preferred embodiment, the cellculture solution comprises an ammonium concentration of no more than 5mM. In another specific embodiment, the ammonium concentration of thecell culture solution is maintained at an ammonium concentration of nomore than 5 mM for at least 7 days. In a preferred embodiment, the cellculture solution comprises an ammonium concentration of no more than 4mM. In another specific embodiment, the ammonium concentration of thecell culture solution is maintained at an ammonium concentration of nomore than 4 mM for at least 7 days. In yet other embodiments, the cellculture media comprises an ammonium concentration of no more than 10 mM,or no more than 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, orless. In yet another embodiment, the ammonium concentration of the cellculture solution is maintained at a low level for the duration of theprocess (i.e., for the entire time the culture is being used to producerVWF). In a preferred embodiment, the recovered supernatant has a rVWFspecific ristocetin cofactor activity of at least 50 mU/μg rVWF. In amore preferred embodiment, the recovered supernatant has a rVWF specificristocetin cofactor activity of at least 70 mU/μg rVWF.

In one embodiment of the method described above, the basal cell culturemedia is supplemented with copper to provide a final copperconcentration of about 4.3 μg/L. In one embodiment, the cell culturesolution further comprises an ammonium concentration of less than 10 mM.In another specific embodiment, the ammonium concentration of the cellculture solution is maintained at an ammonium concentration of no morethan 10 mM for at least 7 days. In a preferred embodiment, the cellculture solution comprises an ammonium concentration of no more than 5mM. In another specific embodiment, the ammonium concentration of thecell culture solution is maintained at an ammonium concentration of nomore than 5 mM for at least 7 days. In a preferred embodiment, the cellculture solution comprises an ammonium concentration of no more than 4mM. In another specific embodiment, the ammonium concentration of thecell culture solution is maintained at an ammonium concentration of nomore than 4 mM for at least 7 days. In yet other embodiments, the cellculture solution comprises an ammonium concentration of no more than 10mM, or no more than 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1mM, or less. In yet another embodiment, the ammonium concentration ofthe cell culture solution is maintained at a low level for the durationof the process (i.e., for the entire time the culture is being used toproduce rVWF). In a preferred embodiment, the recovered supernatant hasa rVWF specific ristocetin cofactor activity of at least 50 mU/μg rVWF.In a more preferred embodiment, the recovered supernatant has a rVWFspecific ristocetin cofactor activity of at least 70 mU/μg rVWF.

In one embodiment of the method described above, the basal cell culturemedia is supplemented with copper to provide a final copperconcentration of between 2 μg/L and 20 μg/L. In one embodiment, the cellculture solution further comprises an ammonium concentration of lessthan 10 mM. In another specific embodiment, the ammonium concentrationof the cell culture solution is maintained at an ammonium concentrationof no more than 10 mM for at least 7 days. In a preferred embodiment,the cell culture solution comprises an ammonium concentration of no morethan 5 mM. In another specific embodiment, the ammonium concentration ofthe cell culture is maintained at an ammonium concentration of no morethan 5 mM for at least 7 days. In a preferred embodiment, the cellculture solution comprises an ammonium concentration of no more than 4mM. In another specific embodiment, the ammonium concentration of thecell culture is maintained at an ammonium concentration of no more than4 mM for at least 7 days. In yet other embodiments, the cell culturesolution comprises an ammonium concentration of no more than 10 mM, orno more than 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, orless. In yet another embodiment, the ammonium concentration of the cellculture is maintained at a low level for the duration of the process(i.e., for the entire time the culture is being used to produce rVWF).In a preferred embodiment, the recovered supernatant has a rVWF specificristocetin cofactor activity of at least 50 mU/μg rVWF. In a morepreferred embodiment, the recovered supernatant has a rVWF specificristocetin cofactor activity of at least 70 mU/μg rVWF.

In one embodiment of the method described above, the basal cell culturemedia is supplemented with copper to provide a final copperconcentration of between 3 μg/L and 10 μg/L. In one embodiment, the cellculture solution further comprises an ammonium concentration of lessthan 10 mM. In another specific embodiment, the ammonium concentrationof the cell culture is maintained at an ammonium concentration of nomore than 10 mM for at least 7 days. In a preferred embodiment, the cellculture solution comprises an ammonium concentration of no more than 5mM. In another specific embodiment, the ammonium concentration of thecell culture solution is maintained at an ammonium concentration of nomore than 5 mM for at least 7 days. In a preferred embodiment, the cellculture solution comprises an ammonium concentration of no more than 4mM. In another specific embodiment, the ammonium concentration of thecell culture is maintained at an ammonium concentration of no more than4 mM for at least 7 days. In yet other embodiments, the cell culturesolution comprises an ammonium concentration of no more than 10 mM, orno more than 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, orless. In yet another embodiment, the ammonium concentration of the cellculture solution is maintained at a low level for the duration of theprocess (i.e., for the entire time the culture is being used to producerVWF). In a preferred embodiment, the recovered supernatant has a rVWFspecific ristocetin cofactor activity of at least 50 mU/μg rVWF. In amore preferred embodiment, the recovered supernatant has a rVWF specificristocetin cofactor activity of at least 70 mU/μg rVWF.

In one embodiment of the method described above, the basal cell culturemedia is supplemented with copper to provide a final copperconcentration of between 4 μg/L and 7.5 μg/L. In one embodiment, thecell culture solution further comprises an ammonium concentration ofless than 10 mM. In another specific embodiment, the ammoniumconcentration of the cell culture is maintained at an ammoniumconcentration of no more than 10 mM for at least 7 days. In a preferredembodiment, the cell culture solution comprises an ammoniumconcentration of no more than 5 mM. In another specific embodiment, theammonium concentration of the cell culture is maintained at an ammoniumconcentration of no more than 5 mM for at least 7 days. In a preferredembodiment, the cell culture solution comprises an ammoniumconcentration of no more than 4 mM. In another specific embodiment, theammonium concentration of the cell culture is maintained at an ammoniumconcentration of no more than 4 mM for at least 7 days. In yet otherembodiments, the cell culture solution comprises an ammoniumconcentration of no more than 10 mM, or no more than 9 mM, 8 mM, 7 mM, 6mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less. In yet another embodiment,the ammonium concentration of the cell culture is maintained at a lowlevel for the duration of the process (i.e., for the entire time theculture is being used to produce rVWF). In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment, the present invention provides a method for producinga recombinant Von Willebrand Factor (rVWF) composition, the methodcomprising the steps of: (a) providing a basal cell culture media; (b)supplementing the basal cell culture media with copper; (c) providingone or more cells comprising a nucleic acid encoding a rVWF protein; (d)culturing the one or more cells in the copper supplemented cell culturemedia such that rVWF is expressed and excreted from the cells into aculture supernatant; and (e) recovering at least a portion of theculture supernatant, wherein the NH₄ ⁺ concentration of the supernatantis maintained at a low level for at least 7 days, and further whereinthe recovered supernatant has a rVWF specific ristocetin cofactoractivity of at least 30 mU/μg rVWF. In a specific embodiment, the copperconcentration and NH₄ ⁺ concentration of the cell culture solution ismaintained at a concentration according to any one of variations 1 to440, as set forth in Table 1. In a preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least50 mU/μg rVWF. In a more preferred embodiment, the recovered supernatanthas a rVWF specific ristocetin cofactor activity of at least 70 mU/μgrVWF.

In one embodiment of the method described above, the copperconcentration and NH₄ ⁺ concentration of the cell culture is maintainedat a concentration according to any one of variations 1 to 440, as setforth in Table 1 for at least 14 days. In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment of the method described above, the copperconcentration and NH₄ ⁺ concentration of the cell culture is maintainedat a concentration according to any one of variations 1 to 440, as setforth in Table 1 for at least 21 days. In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment of the method described above, the copperconcentration and NH₄ ⁺ concentration of the cell culture is maintainedat a concentration according to any one of variations 1 to 440, as setforth in Table 1 for at least 28 days. In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment of the method described above, the copperconcentration and NH₄ ⁺ concentration of the cell culture is maintainedat a concentration according to any one of variations 1 to 440, as setforth in Table 1 for at least 5 weeks. In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment of the method described above, the copperconcentration and NH₄ ⁺ concentration of the cell culture is maintainedat a concentration according to any one of variations 1 to 440, as setforth in Table 1 for at least 6 weeks. In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment of the method described above, the copperconcentration and NH₄ ⁺ concentration of the cell culture is maintainedat a concentration according to any one of variations 1 to 440, as setforth in Table 1 for at least 7 weeks. In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment of the method described above, the copperconcentration and NH₄ ⁺ concentration of the cell culture is maintainedat a concentration according to any one of variations 1 to 440, as setforth in Table 1 for at least 8 weeks. In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment of the method described above, the copperconcentration and NH₄ ⁺ concentration of the cell culture is maintainedat a concentration according to any one of variations 1 to 440, as setforth in Table 1 for at least 9 weeks. In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment, the present invention provides a method for producinga recombinant Von Willebrand Factor (rVWF) composition, the methodcomprising the steps of: (a) providing a basal cell culture media; (b)supplementing the basal cell culture media with copper to provide afinal copper concentration of at least 2.0 μg/L; (c) providing one ormore cells comprising a nucleic acid encoding a rVWF protein; (d)culturing the one or more cells in the copper supplemented cell culturemedia such that rVWF is expressed and excreted from the cells into aculture supernatant; (e) monitoring the ammonium concentration of theculture supernatant; and (f) recovering at least a portion of theculture supernatant, wherein culture supernatant comprising an ammoniumconcentration of more than 10 mM is not used for producing the rVWFcomposition, and further wherein the recovered supernatant has a rVWFspecific ristocetin cofactor activity of at least 30 mU/μg rVWF. Incertain embodiments, the final copper concentration of the supplementedbasal culture media is at least 2.4 μg/L, 3 μg/L, 4 μg/L, 5 μg/L, 6μg/L, 7 μg/L, 8 μg/L, 9 μg/L, 10 μg/L, 15 μg/L, 20 μg/L, or higher. Inother embodiments, the final copper concentration of the supplementedbasal culture media is between 2-20 μg/L, 2-10 μg/L, 3-8 μg/L, or 4-6μg/L. In a preferred embodiment, the recovered supernatant has a rVWFspecific ristocetin cofactor activity of at least 50 mU/μg rVWF. In amore preferred embodiment, the recovered supernatant has a rVWF specificristocetin cofactor activity of at least 70 mU/μg rVWF.

In one embodiment of the method described above, culture supernatantcomprising an ammonium concentration of more than 6 mM is not used forproducing the rVWF composition. In certain embodiments, the final copperconcentration of the supplemented basal culture media is at least 2.4μg/L, 3 μg/L, 4 μg/L, 5 μg/L, 6 μg/L, 7 μg/L, 8 μg/L, 9 μg/L, 10 μg/L,15 μg/L, 20 μg/L, or higher. In other embodiments, the final copperconcentration of the supplemented basal culture media is between 2-20μg/L, 2-10 μg/L, 3-8 μg/L, or 4-6 μg/L. In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment of the method described above, culture supernatantcomprising an ammonium concentration of more than 5 mM is not used forproducing the rVWF composition. In certain embodiments, the final copperconcentration of the supplemented basal culture media is at least 2.4μg/L, 3 μg/L, 4 μg/L, 5 μg/L, 6 μg/L, 7 μg/L, 8 μg/L, 9 μg/L, 10 μg/L,15 μg/L, 20 μg/L, or higher. In other embodiments, the final copperconcentration of the supplemented basal culture media is between 2-20μg/L, 2-10 μg/L, 3-8 μg/L, or 4-6 μg/L. In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

In one embodiment of the method described above, culture supernatantcomprising an ammonium concentration of more than 4 mM is not used forproducing the rVWF composition. In certain embodiments, the final copperconcentration of the supplemented basal culture media is at least 2.4μg/L, 3 μg/L, 4 μg/L, 5 μg/L, 6 μg/L, 7 μg/L, 8 μg/L, 9 μg/L, 10 μg/L,15 μg/L, 20 μg/L, or higher. In other embodiments, the final copperconcentration of the supplemented basal culture media is between 2-20μg/L, 2-10 μg/L, 3-8 μg/L, or 4-6 μg/L. In a preferred embodiment, therecovered supernatant has a rVWF specific ristocetin cofactor activityof at least 50 mU/μg rVWF. In a more preferred embodiment, the recoveredsupernatant has a rVWF specific ristocetin cofactor activity of at least70 mU/μg rVWF.

Recombinant vWF can be produced by expression in a suitable eukaryotichost system. Examples of eukaryotic cells include, without limitation,mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep, and HepG2;insect cells, e.g., SF9 cells, SF21 cells, S2 cells, and High Fivecells; and yeast cells, e.g., Saccharomyces or Schizosaccharomycescells. In one embodiment, the vWF can be expressed in yeast cells,insect cells, avian cells, mammalian cells, and the like. For example,in a human cell line, a hamster cell line, or a murine cell line. In oneparticular embodiment, the cell line is a CHO, BHK, or HEK cell line.Typically, mammalian cells, e.g., CHO cells from a continuous cell line,can be used to express the vWF of the present invention.

In certain embodiments, the nucleic acid sequence comprising a sequencecoding for vWF can be a vector. The vector can be delivered by a virusor can be a plasmid. The nucleic acid sequence coding for the proteincan be a specific gene or a biologically functional part thereof. In oneembodiment, the protein is at least a biologically active part of vWF.

A wide variety of vectors can be used for the expression of the vWF andcan be selected from eukaryotic expression vectors. Examples of vectorsfor eukaryotic expression include: (i) for expression in yeast, vectorssuch as pAO, pPIC, pYES, pMET, using promoters such as AOX1, GAP, GAL1,AUG1, etc; (ii) for expression in insect cells, vectors such as pMT,pAc5, pIB, pMIB, pBAC, etc., using promoters such as PH, p10, MT, Ac5,OpIE2, gp64, polh, etc., and (iii) for expression in mammalian cells,vectors such as pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., and vectorsderived from viral systems such as vaccinia virus, adeno-associatedviruses, herpes viruses, retroviruses, etc., using promoters such asCMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin. An exemplary vectorfor expressing rVWF is described by Kaufman et al. (Mol Cell Biol. 1989March; 9(3):1233-42), the content of which is hereby incorporated byreference in its entirety for all purposes.

In some embodiments of the present invention, the nucleic acid sequencefurther comprises other sequences suitable for a controlled expressionof a protein such as promoter sequences, enhancers, TATA boxes,transcription initiation sites, polylinkers, restriction sites,poly-A-sequences, protein processing sequences, selection markers, andthe like which are generally known to a person of ordinary skill in theart.

In addition to cell culture media comprising an increased copperconcentration, the cell culture conditions of the present invention caninclude an ammonium concentration of less than about 25 mM throughout anentire upstream process in culture systems. In one embodiment, the cellculture conditions include an ammonium concentration of less than about25 mM, in another embodiment less than about 20 mM, in yet anotherembodiment less than about 15 mM, in yet another embodiment less thanabout 10 mM, and in a further embodiment less than about 5 mM.

In some embodiments, the ammonium concentrations of the presentinvention are kept constant throughout the entire upstream process ofthe cell culture system. The cells used according to the presentinvention can be cultivated, e.g., by methods that are modified ascompared to conventional batch-cultivation and feed-batch-cultivation,each of which are generally known in the field. However, suchconventional techniques can produce high concentrations of ammonium atthe end of the culture. The methods of the present invention overcomethis problem by employing production systems that can provide acontinuous supply of culture media through techniques such as, e.g.,perfusion or chemostat cultures. Following culture of the host cells,the vWF can be recovered from the spent medium using standardmethodologies, such as ultrafiltration or centrifugation. If desired,the vWF can be purified by, e.g., ion exchange and/or size exclusionchromatography and the like.

A continuous culture (e.g., a perfusion or chemostat culture) can be asuspension culture that is continuously supplied with nutrients by theinflow of fresh medium, wherein the culture volume is usually constant.Similarly, continuous fermentation can refer to a process in which cellsor micro-organisms are maintained in culture in the exponential growthphase by the continuous addition of fresh medium that is exactlybalanced by the removal of cell suspension from the bioreactor.Furthermore, a stirred-tank reactor system can be used for suspension,perfusion, chemostatic, and/or microcarrier cultures. Generally, thestirred-tank reactor system can be operated as any conventionalstirred-tank reactor with any type of agitator such as a Rushton,hydrofoil, pitched blade, or marine.

C. Methods of Producing Recombinant ADAMTS13 (A13)

In another aspect, the present invention further relates to methods forproducing rA13 under cell culture conditions comprising a cell culturemedium having an increased copper concentration. In certain embodiments,the culture also comprises a low ammonium concentration.

In one embodiment, the present invention provides a method for producinga recombinant ADAMTS13 (rA13) composition, the method comprising thesteps of: (a) providing a basal cell culture media; (b) supplementingthe basal cell culture media with copper to provide a final copperconcentration of at least 1.0 μg/L; (c) providing one or more cellscomprising a nucleic acid encoding a rA13 protein; (d) culturing the oneor more cells in the copper supplemented cell culture media such thatrA13 is expressed and excreted from the cells into a culturesupernatant; and (e) recovering at least a portion of the culturesupernatant, wherein at least 1500 Units FRETS-VWF73 activity per litersupplemented basal cell culture media per day (i.e., 1500 UnitsFRETS-VWF73 activity per day per liter cell culture; P FRETS) is presentin the recovered culture supernatant. In certain embodiments, the finalcopper concentration of the supplemented basal culture media is at least2 μg/L, 3 μg/L, 4 μg/L, 5 μg/L, 6 μg/L, or higher. In other embodiments,the final copper concentration of the supplemented basal culture mediais between 1-6 μg/L, 2-5 μg/L, 2-4 μg/L, or 3-4 μg/L. In a preferredembodiment, at least 2000 Units FRETS-VWF73 activity per litersupplemented basal cell culture media per day is present in therecovered culture supernatant. In a more preferred embodiment, at least2500 Units FRETS-VWF73 activity per liter supplemented basal cellculture media per day is present in the recovered culture supernatant.In a most preferred embodiment, at least 3000 Units FRETS-VWF73 activityper liter supplemented basal cell culture media per day is present inthe recovered culture supernatant. In certain embodiments, the methodsprovide sustainably improved volumetric FRETS-VWF73 productivity (PFRETS). For example, in certain embodiments, at least 1500 UnitsFRETS-VWF73 activity per liter supplemented basal cell culture media isrecovered daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49,56, 63, 70, or more days. In a preferred embodiment, at least 2000 UnitsFRETS-VWF73 activity per liter supplemented basal cell culture media isrecovered daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49,56, 63, 70, or more days. In a more preferred embodiment, at least 2500Units FRETS-VWF73 activity per liter supplemented basal cell culturemedia is recovered daily for at least 7 days, or at least 14, 21, 28,35, 42, 49, 56, 63, 70, or more days. In a most preferred embodiment, atleast 3000 Units FRETS-VWF73 activity per liter supplemented basal cellculture media is recovered daily for at least 7 days, or at least 14,21, 28, 35, 42, 49, 56, 63, 70, or more days. In a specific embodiment,the cell density of the continuous cell culture for production of rA13is maintained at a concentration of no more than 4.0×10⁶ cells/mL for anextended period. In other specific embodiments, the cell density ismaintained at no more than 3.5×10⁶ cells/mL, 3.0×10⁶ cells/mL, 2.5×10⁶cells/mL, 2.0×10⁶ cells/mL, 1.5×10⁶ cells/mL, 1.0×10⁶ cells/mL, or less.In one embodiment, the cell density is maintained at between 3.0×10⁶cells/mL and 4.0×10⁶ cells/mL.

In one embodiment of the methods described above, the recoveredsupernatant has at least 4 Units FRETS-VWF73 activity per mL supernatant(FRETS). In a preferred embodiment, the recovered supernatant has atleast 6 Units FRETS-VWF73 activity per mL supernatant. In a morepreferred embodiment, the recovered supernatant has at least 8 UnitsFRETS-VWF73 activity per mL supernatant. In a most preferred embodiment,the recovered supernatant has at least 10 Units FRETS-VWF73 activity permL supernatant. In certain embodiments, the methods provide sustainablyimproved FRETS production. For example, in certain embodiments,supernatant having at least 4 Units FRETS-VWF73 activity per mL arerecovered daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49,56, 63, 70, or more days. In a preferred embodiment, supernatant havingat least 6 Units FRETS-VWF73 activity per mL are recovered daily for atleast 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70, or moredays. In a more preferred embodiment, supernatant having at least 8Units FRETS-VWF73 activity per mL are recovered daily for at least 7days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70, or more days. In amost preferred embodiment, supernatant having at least 10 UnitsFRETS-VWF73 activity per mL are recovered daily for at least 7 days, orat least 14, 21, 28, 35, 42, 49, 56, 63, 70, or more days.

In one embodiment of the methods described above, the cell cultureresults in the production of at least 800 mU of FRETS-VWF73 activity per10⁶ cells present in the culture per day (i.e., q FRETS). In a preferredembodiment, the cell culture results in the production of at least 1 Uof FRETS-VWF73 activity per 10⁶ cells present in the culture per day. Ina more preferred embodiment, the cell culture results in the productionof at least 1.2 U of FRETS-VWF73 activity per 10⁶ cells present in theculture per day. In a most preferred embodiment, the cell cultureresults in the production of at least 1.4 U of FRETS-VWF73 activity per10⁶ cells present in the culture per day. In certain embodiments, themethods provide sustainably improved cell-specific FRETS-VWF73productivity (q FRETS). For example, in certain embodiments, the cellculture results in the production of at least 800 mU of FRETS-VWF73activity per 10⁶ cells present in the culture daily for at least 7 days,or at least 14, 21, 28, 35, 42, 49, 56, 63, 70, or more days. In apreferred embodiment, the cell culture results in the production of atleast 1 U of FRETS-VWF73 activity per 10⁶ cells present in the culturedaily for at least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63,70, or more days. In a more preferred embodiment, the cell cultureresults in the production of at least 1.2 U of FRETS-VWF73 activity per10⁶ cells present in the culture daily for at least 7 days, or at least14, 21, 28, 35, 42, 49, 56, 63, 70, or more days. In a most preferredembodiment, the cell culture results in the production of at least 1.4 Uof FRETS-VWF73 activity per 10⁶ cells present in the culture daily forat least 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70, or moredays.

In one embodiment of the methods described above, the cell cultureresults in the production of at least 1 mg rA13, as measured by ELISA,per liter culture per day (P ELISA). In a preferred embodiment, the cellculture results in the production of at least 1.5 mg rA13, as measuredby ELISA, per liter culture per day. In a more preferred embodiment, thecell culture results in the production of at least 2 mg rA13, asmeasured by ELISA, per liter culture per day. In certain embodiments,the methods provide sustainably improved rA13 production. For example,in certain embodiments, the cell culture results in the production of atleast 1 mg rA13, as measured by ELISA, per liter culture daily for atleast 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70, or moredays. In a preferred embodiment, the cell culture results in theproduction of at least 1.5 mg rA13, as measured by ELISA, per literculture daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49,56, 63, 70, or more days. In a more preferred embodiment, the cellculture results in the production of at least 2 mg rA13, as measured byELISA, per liter culture daily for at least 7 days, or at least 14, 21,28, 35, 42, 49, 56, 63, 70, or more days.

In one embodiment of the methods described above, the cell cultureresults in the production of at least 0.5 μg rA13, as measured by ELISA,per 10⁶ cells present in the culture per day (i.e., q ELISA). In apreferred embodiment, the cell culture results in the production of atleast 0.7 μg rA13, as measured by ELISA, per 10⁶ cells present in theculture per day. In a more preferred embodiment, the cell cultureresults in the production of at least 0.9 μg rA13, as measured by ELISA,per 10⁶ cells present in the culture per day. In certain embodiments,the methods provide sustainably improved q ELISA production. Forexample, in certain embodiments, the cell culture results in theproduction of at least 0.5 μg rA13, as measured by ELISA, per 10⁶ cellspresent in the culture daily for at least 7 days, or at least 14, 21,28, 35, 42, 49, 56, 63, 70, or more days. In a preferred embodiment, thecell culture results in the production of at least 0.7 μg rA13, asmeasured by ELISA, per 10⁶ cells present in the culture daily for atleast 7 days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70, or moredays. In a more preferred embodiment, the cell culture results in theproduction of at least 0.9 μg rA13, as measured by ELISA, per 10⁶ cellspresent in the culture daily for at least 7 days, or at least 14, 21,28, 35, 42, 49, 56, 63, 70, or more days.

In one embodiment of the methods described above, the recoveredsupernatant has at least 3 μg rA13, as measured by ELISA, per mLsupernatant. In a preferred embodiment, the recovered supernatant has atleast 4 μg rA13, as measured by ELISA, per mL supernatant. In a morepreferred embodiment, the recovered supernatant has at least 5 μg rA13,as measured by ELISA, per mL supernatant. In a most preferredembodiment, the recovered supernatant has at least 6 μg rA13, asmeasured by ELISA, per mL supernatant. In certain embodiments, themethods provide sustainably improved rA13 production. For example, incertain embodiments, supernatant having at least 3 μg rA13 per mL arerecovered daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49,56, 63, 70, or more days. In a preferred embodiment, supernatant havingat least 4 μg rA13 per mL are recovered daily for at least 7 days, or atleast 14, 21, 28, 35, 42, 49, 56, 63, 70, or more days. In a morepreferred embodiment, supernatant having at least 5 μg rA13 per mL arerecovered daily for at least 7 days, or at least 14, 21, 28, 35, 42, 49,56, 63, 70, or more days. In a most preferred embodiment, supernatanthaving at least 6 μg rA13 per mL are recovered daily for at least 7days, or at least 14, 21, 28, 35, 42, 49, 56, 63, 70, or more days.

In one embodiment of the methods described above, the cell culturesolution further comprises an ammonium concentration of less than 10 mM.In another specific embodiment, the ammonium concentration of the cellculture is maintained at an ammonium concentration of no more than 10 mMfor at least 7 days. In a preferred embodiment, the cell culturesolution comprises an ammonium concentration of no more than 5 mM. Inanother specific embodiment, the ammonium concentration of the cellculture is maintained at an ammonium concentration of no more than 5 mMfor at least 7 days. In a preferred embodiment, the cell culturesolution comprises an ammonium concentration of no more than 4 mM. Inanother specific embodiment, the ammonium concentration of the cellculture is maintained at an ammonium concentration of no more than 4 mMfor at least 7 days. In yet other embodiments, the cell culture solutioncomprises an ammonium concentration of no more than 10 mM, or no morethan 9 mM, 8 mM, 7 mM, 6 mM, 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, or less. Inyet another embodiment, the ammonium concentration of the cell cultureis maintained at a low level for the duration of the process (i.e., forthe entire time the culture is being used to produce rA13). In aparticular embodiment, the culture solution has a copper and ammoniumconcentration according to any one of variations 1 to 440, as providedin Table 1.

In one embodiment, the present invention provides a method for producinga recombinant ADAMTS13 (rA13) composition, the method comprising thesteps of: (a) providing a basal cell culture media; (b) supplementingthe basal cell culture media with copper and zinc; (c) providing one ormore cells comprising a nucleic acid encoding a rA13 protein; (d)culturing the one or more cells in the copper supplemented cell culturemedia such that rA13 is expressed and excreted from the cells into aculture supernatant; and (e) recovering at least a portion of theculture supernatant, wherein at least 1500 Units FRETS-VWF73 activityper liter supplemented basal cell culture media per day is present inthe recovered culture supernatant. In one embodiment, the culture mediumcontains at least 1 μg/L copper and at least 2 μM zinc. In otherembodiments, the media contains at least 2 μg/L copper or at least 4μg/L copper. In one embodiment wherein the media is supplemented withcopper, the culture medium also contains at least at or about 5 μM zinc.In one embodiment, the culture medium also contains at or about between2 μM and 12 μM zinc. In another embodiment, the culture medium alsocontains at or about between 5 μM and 12 μM zinc. In yet otherembodiments, the culture medium also may contain at least at or about 2μM, or at least at or about 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 20 μM, 25 μM, 30 μM, or morezinc. In one embodiment, the culture medium contains a copper and zincconcentration according to any one of variations 441 to 880, as setforth in Table 2. In a preferred embodiment, at least 2000 UnitsFRETS-VWF73 activity per liter supplemented basal cell culture media perday is present in the recovered culture supernatant. In a more preferredembodiment, at least 2500 Units FRETS-VWF73 activity per litersupplemented basal cell culture media per day is present in therecovered culture supernatant. In a most preferred embodiment, at least3000 Units FRETS-VWF73 activity per liter supplemented basal cellculture media per day is present in the recovered culture supernatant.

In one embodiment, the present invention provides a method for producinga recombinant ADAMTS13 (rA13) composition, the method comprising thesteps of: (a) providing a basal cell culture media; (b) supplementingthe basal cell culture media with copper and calcium; (c) providing oneor more cells comprising a nucleic acid encoding a rA13 protein; (d)culturing the one or more cells in the copper supplemented cell culturemedia such that rA13 is expressed and excreted from the cells into aculture supernatant; and (e) recovering at least a portion of theculture supernatant, wherein at least 1500 Units FRETS-VWF73 activityper liter supplemented basal cell culture media per day is present inthe recovered culture supernatant. In one embodiment, the culture mediumcontains at least 1 μg/L copper and at least 0.5 mM calcium. In otherembodiments, the media contains at least 2 μg/L copper or at least 4μg/L copper. In another embodiment wherein the media is supplementedwith copper, the culture medium also contains at least 1.5 mM calcium.In one embodiment, the culture medium contains at or about between 0.5mM and 1.5 mM calcium. In yet other embodiments, the culture medium maycontain at least at or about 0.5 mM, or at least at or about 0.6 mM, 0.7mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM,3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, or more calcium. In one embodiment, theculture medium contains a copper and calcium concentration according toany one of variations 881 to 1320, as set forth in Table 3. In apreferred embodiment, at least 2000 Units FRETS-VWF73 activity per litersupplemented basal cell culture media per day is present in therecovered culture supernatant. In a more preferred embodiment, at least2500 Units FRETS-VWF73 activity per liter supplemented basal cellculture media per day is present in the recovered culture supernatant.In a most preferred embodiment, at least 3000 Units FRETS-VWF73 activityper liter supplemented basal cell culture media per day is present inthe recovered culture supernatant.

In one embodiment, the present invention provides a method for producinga recombinant ADAMTS13 (rA13) composition, the method comprising thesteps of: (a) providing a basal cell culture media; (b) supplementingthe basal cell culture media with copper, zinc, and calcium; (c)providing one or more cells comprising a nucleic acid encoding a rA13protein; (d) culturing the one or more cells in the copper supplementedcell culture media such that rA13 is expressed and excreted from thecells into a culture supernatant; and (e) recovering at least a portionof the culture supernatant, wherein at least 1500 Units FRETS-VWF73activity per liter supplemented basal cell culture media per day ispresent in the recovered culture supernatant. In one embodiment, theculture medium has a calcium concentration of at least 0.5 mM and acopper and zinc concentration according to any one of variations 441 to880, as set forth in Table 2. In another specific embodiment, theculture medium has a calcium concentration of at least 1.5 mM and acopper and zinc concentration according to any one of variations 441 to880, as set forth in Table 2. In another specific embodiment, theculture medium has a calcium concentration between 0.5 mM and 1.5 mM anda copper and zinc concentration according to any one of variations 441to 880, as set forth in Table 2. In yet other embodiments, the culturemedium has a calcium concentration of at least 0.6 mM, 0.7 mM, 0.8 mM,0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM,1.8 mM, 1.9 mM, 2.0 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3.0 mM, 3.5 mM, 4.0mM, 4.5 mM, 5.0 mM, or more, and a copper and zinc concentrationaccording to any one of variations 441 to 880, as set forth in Table 2.In a preferred embodiment, at least 2000 Units FRETS-VWF73 activity perliter supplemented basal cell culture media per day is present in therecovered culture supernatant. In a more preferred embodiment, at least2500 Units FRETS-VWF73 activity per liter supplemented basal cellculture media per day is present in the recovered culture supernatant.In a most preferred embodiment, at least 3000 Units FRETS-VWF73 activityper liter supplemented basal cell culture media per day is present inthe recovered culture supernatant.

In one embodiment, the present invention provides a method for producinga recombinant ADAMTS13 (rA13) composition, the method comprising thesteps of: (a) providing a basal cell culture media; (b) supplementingthe basal cell culture media with copper and nicotinamide; (c) providingone or more cells comprising a nucleic acid encoding a rA13 protein; (d)culturing the one or more cells in the copper supplemented cell culturemedia such that rA13 is expressed and excreted from the cells into aculture supernatant; and (e) recovering at least a portion of theculture supernatant, wherein at least 1500 Units FRETS-VWF73 activityper liter supplemented basal cell culture media per day is present inthe recovered culture supernatant. In one embodiment, the culture mediumcontains at least 1 μg/L copper and at least 2 mg/L nicotinamide(vitamin B3). In other embodiments, the media contains at least 2 μg/Lcopper or at least 4 μg/L copper. In another embodiment wherein themedia is supplemented with copper, the culture medium also contains atleast 7 mg/L nicotinamide (vitamin B3). In one embodiment, the culturemedium contains at or about between 2 mg/L and 10 mg/L nicotinamide(vitamin B3). In yet other embodiments, the culture medium may containat least at or about 2 mg/L, 3 mg/L, 4 mg/L, 5 mg/L, 6 mg/L, 7 mg/L, 8mg/L, 9 mg/L, 10 mg/L, 15 mg/L, 20 mg/L, or higher concentrations ofnicotinamide (vitamin B3). In one embodiment, the culture mediumcontains a copper and nicotinamide concentration according to any one ofvariations 1321 to 1760, as set forth in Table 4. In a preferredembodiment, at least 2000 Units FRETS-VWF73 activity per litersupplemented basal cell culture media per day is present in therecovered culture supernatant. In a more preferred embodiment, at least2500 Units FRETS-VWF73 activity per liter supplemented basal cellculture media per day is present in the recovered culture supernatant.In a most preferred embodiment, at least 3000 Units FRETS-VWF73 activityper liter supplemented basal cell culture media per day is present inthe recovered culture supernatant.

In one embodiment, the present invention provides a method for producinga recombinant ADAMTS13 (rA13) composition, the method comprising thesteps of: (a) providing a basal cell culture media; (b) supplementingthe basal cell culture media with copper, zinc, and nicotinamide; (c)providing one or more cells comprising a nucleic acid encoding a rA13protein; (d) culturing the one or more cells in the copper supplementedcell culture media such that rA13 is expressed and excreted from thecells into a culture supernatant; and (e) recovering at least a portionof the culture supernatant, wherein at least 1500 Units FRETS-VWF73activity per liter supplemented basal cell culture media per day ispresent in the recovered culture supernatant. In one embodiment, thecell culture medium has a nicotinamide concentration of at least 2 mg/mLand a copper and zinc concentration according to any one of variations441 to 880, as set forth in Table 2. In another specific embodiment, theculture medium has a nicotinamide concentration of at least 7 mg/mL mMand a copper and zinc concentration according to any one of variations441 to 880, as set forth in Table 2. In another specific embodiment, theculture medium has a nicotinamide concentration between 2 mg/mL and 10mg/mL and a copper and zinc concentration according to any one ofvariations 441 to 880, as set forth in Table 2. In yet otherembodiments, the culture medium has a nicotinamide concentration of atleast 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, ormore, and a copper and zinc concentration according to any one ofvariations 441 to 880, as set forth in Table 2. In a preferredembodiment, at least 2000 Units FRETS-VWF73 activity per litersupplemented basal cell culture media per day is present in therecovered culture supernatant. In a more preferred embodiment, at least2500 Units FRETS-VWF73 activity per liter supplemented basal cellculture media per day is present in the recovered culture supernatant.In a most preferred embodiment, at least 3000 Units FRETS-VWF73 activityper liter supplemented basal cell culture media per day is present inthe recovered culture supernatant.

In one embodiment, the present invention provides a method for producinga recombinant ADAMTS13 (rA13) composition, the method comprising thesteps of: (a) providing a basal cell culture media; (b) supplementingthe basal cell culture media with copper, calcium, and nicotinamide; (c)providing one or more cells comprising a nucleic acid encoding a rA13protein; (d) culturing the one or more cells in the copper supplementedcell culture media such that rA13 is expressed and excreted from thecells into a culture supernatant; and (e) recovering at least a portionof the culture supernatant, wherein at least 1500 Units FRETS-VWF73activity per liter supplemented basal cell culture media per day ispresent in the recovered culture supernatant. In one embodiment, thecell culture medium has a nicotinamide concentration of at least 2 mg/mLand a copper and calcium concentration according to any one ofvariations 881 to 1320, as set forth in Table 3. In another specificembodiment, the culture medium has a nicotinamide concentration of atleast 7 mg/mL mM and a copper and calcium concentration according to anyone of variations 881 to 1320, as set forth in Table 3. In anotherspecific embodiment, the culture medium has a nicotinamide concentrationbetween 2 mg/mL and 10 mg/mL and a copper and calcium concentrationaccording to any one of variations 881 to 1320, as set forth in Table 3.In yet other embodiments, the culture medium has a nicotinamideconcentration of at least 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14mg/mL, 15 mg/mL, or more, and a copper and calcium concentrationaccording to any one of variations 881 to 1320, as set forth in Table 3.In a preferred embodiment, at least 2000 Units FRETS-VWF73 activity perliter supplemented basal cell culture media per day is present in therecovered culture supernatant. In a more preferred embodiment, at least2500 Units FRETS-VWF73 activity per liter supplemented basal cellculture media per day is present in the recovered culture supernatant.In a most preferred embodiment, at least 3000 Units FRETS-VWF73 activityper liter supplemented basal cell culture media per day is present inthe recovered culture supernatant.

Recombinant ADAMTS proteins can be produced by expression in anysuitable prokaryotic or eukaryotic host system. Examples of eukaryoticcells include, without limitation, mammalian cells, such as CHO, COS,HEK 293, BHK, SK-Hep, and HepG2; insect cells, for example SF9 cells,SF21 cells, S2 cells, and High Five cells; and yeast cells, for exampleSaccharomyces or Schizosaccharomyces cells. In one embodiment, theADAMTS proteins can be expressed in bacterial cells, yeast cells, insectcells, avian cells, mammalian cells, and the like. For example, in ahuman cell line, a hamster cell line, or a murine cell line. In oneparticular embodiment, the cell line is a CHO, BHK, or HEK cell line. Ina preferred embodiment, the cell line is a CHO cell line. In a specificembodiment, CHO clones capable of stably expressing rA13 are prepared byco-transfecting a CHO cell with coding sequences for rA13 and adihydrofolate reductase (e.g., a murine dhfr gene) and selecting forgrowth in the presence of increasing levels of methotrexate.

In one embodiment, the cells may be any mammalian cell that can becultured, preferably in a manufacturing process (i.e., at least 10liter, preferrably at least 100 liters), to produce a desired ADAMTSprotein such as ADAMTS13. Examples include the monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); Chinese hamster ovary cells/-DHFR, such as the DUKX-B11subclone (CHO, Uriaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216(1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod, 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci.,383:44-68 (1982)); MRC 5 cells; FS4 cells; and the human hepatoma line(Hep G2). Preferably, the cell line is a rodent cell line, especially ahamster cell line such as CHO or BHK.

A wide variety of vectors can be used for the expression of an ADAMTSprotein (e.g., ADAMTS13) and can be selected from eukaryotic andprokaryotic expression vectors. In certain embodiments, a plasmid vectoris contemplated for use in expressing an ADAMTS protein (e.g.,ADAMTS13). In general, plasmid vectors containing replicon and controlsequences which are derived from species compatible with the host cellare used in connection with these hosts. The vector can carry areplication site, as well as marking sequences which are capable ofproviding phenotypic selection in transformed cells. The plasmid willcomprise a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) operable linked to one or more control sequences, for example,a promoter.

A preferred method of preparing stable CHO cell clones expressing arecombinant ADAMTS protein is as follows. A DHFR deficient CHO cell lineDUKX-B 11 is transfected with a DHFR expression vector to allow forexpression of the relevant recombinant protein. An exemplary method isdescribed by Plaimauer et al. (Blood. 2002 Nov. 15; 100(10):3626-32.Epub 2002 Jul. 12), the content of which is hereby incorporated byreference in its entirety for all purposes. Selection is carried out bygrowth in Hypoxanthine/Thymidine (HT) free media and amplification ofthe relevant region coding for expression of the recombinant ADAMTSprotein and DHFR gene is achieved by propagation of the cells inincreasing concentrations of methotrexate. Where appropriate, CHO celllines may be adapted for growth in serum and/or protein free medium,essentially as described in U.S. Pat. No. 6,100,061 (Reiter et al.,lmmuno Aktiengesellschaft).

In another preferred embodiment, stable HEK293 cells are prepared bytransfecting with a construct containing a hygromycin selectable markerand selecting transformants by antibiotic resistance.

The ability of certain viruses to infect cells or enter cells viareceptor-mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Accordingly, in certain embodiments, a viral vector isused to introduce a nucleotide sequence encoding an ADAMTS protein(e.g., ADAMTS13) into a host cell for expression. The viral vector willcomprise a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) operable linked to one or more control sequences, for example,a promoter. Alternatively, the viral vector may not contain a controlsequence and will instead rely on a control sequence within the hostcell to drive expression of the ADAMTS protein. Non-limiting examples ofvirus vectors that may be used to deliver a nucleic acid includeAdenoviral vectors, AAV vectors, and Retroviral vectors.

In one embodiment, an Adenovirus expression vector include thoseconstructs containing adenovirus sequences sufficient to supportpackaging of the construct and to ultimately express an ADAMTS constructthat has been cloned therein. Adenoviral vectors allow for theintroduction of foreign sequences up to 7 kb (Grunhaus et al., Seminarin Virology, 200(2):535-546, 1992)).

In another embodiment, an adeno-associated virus (AAV) can be used tointroduce a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) into a host cell for expression. AAV systems have beendescribed previously and are generally well known in the art (Kelleherand Vos, Biotechniques, 17(6):1110-7, 1994; Cotten et al., Proc NatlAcad Sci USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3):141-64,1994; Muzyczka, Curr Top Microbiol Immunol, 158:97-129, 1992). Detailsconcerning the generation and use of rAAV vectors are described, forexample, in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporatedherein by reference in their entireties for all purposes.

In one embodiment, a retroviral expression vector can be used tointroduce a nucleotide sequence encoding an ADAMTS protein (e.g.,ADAMTS13) into a host cell for expression. These systems have beendescribed previously and are generally well known in the art (Mann etal., Cell, 33:153-159, 1983; Nicolas and Rubinstein, In: Vectors: Asurvey of molecular cloning vectors and their uses, Rodriguez andDenhardt, eds., Stoneham: Butterworth, pp. 494-513, 1988; Temin, In:Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188,1986). In a specific embodiment, the retroviral vector is a lentiviralvector (see, for example, Naldini et al., Science, 272(5259):263-267,1996; Zufferey et al., Nat Biotechnol, 15(9):871-875, 1997; Blomer etal., J Virol., 71(9):6641-6649, 1997; U.S. Pat. Nos. 6,013,516 and5,994,136).

Non-limiting examples of vectors for prokaryotic expression includeplasmids such as pRSET, pET, pBAD, etc., wherein the promoters used inprokaryotic expression vectors include lac, trc, trp, recA, araBAD, etc.Examples of vectors for eukaryotic expression include: (i) forexpression in yeast, vectors such as pAO, pPIC, pYES, pMET, usingpromoters such as AOX1, GAP, GAL1, AUG1, etc; (ii) for expression ininsect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., usingpromoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc., and (iii)for expression in mammalian cells, vectors such as pSVL, pCMV, pRc/RSV,pcDNA3, pBPV, etc., and vectors derived form viral systems such asvaccinia virus, adeno-associated viruses, herpes viruses, retroviruses,etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, andβ-actin. An exemplary vector for expressing rA13 is described byPlaimauer et al. (Blood. 2002 Nov 15;100(10):3626-32. Epub 2002 Jul.12), the content of which is hereby incorporated by reference in itsentirety for all purposes.

In certain embodiments, the cell-culture methods of the invention maycomprise the use of a microcarrier. The present invention provides,among other aspect, methods of large-scale ADAMTS protein expression. Insome embodiments, the cell-cultures of the embodiments can be performedin large bioreactors under conditions suitable for providing highvolume-specific culture surface areas to achieve high cell densities andprotein expression. One means for providing such growth conditions is touse microcarriers for cell-culture in stirred tank bioreactors. Inanother embodiment, these growth requirements are met via the use of asuspension cell culture.

V. SPECIFIC EMBODIMENTS

A. Recombinant Von Willebrand Factor (rVWF)

Recombinant vWF can be expressed in mammalian cells, but the specificactivity of the vWF can vary widely depending on the cell cultureconditions and has not been shown to be comparable or equal to that ofvWF isolated from blood plasma. The present invention is based in-parton the surprising result that cell culture media having at least 2.4μg/L of copper provides an advantageous effect of promoting expressionof high molecular weight vWF having a high specific activity. Inparticular, the high molecular weight, recombinant vWF of the presentinvention can include a highly multimeric form comprising about 14 toabout 22 dimers and a specific Ristocetin activity of at least about 30mU/μg. The cell culture processes according to the present inventionalso allow for maintaining low NH4⁺ levels (e.g., less than 10 mM)during the upstream process in cell culture systems, thereby reducingdeleterious effects to post-translational modifications. It is believedthat the present invention provides for the first time cell cultureconditions comprising a medium having a suitable copper concentration incombination with appropriate levels of ammonium in the supernatant toexpress a highly multimeric vWF with a high specific activity.

In one aspect, the present invention relates to cell culture conditionsfor producing recombinant, high molecular weight vWF with a highspecific activity. The cell culture conditions of the present inventioncan include, for example, a cell culture medium with an increased copperconcentration and/or cell culture supernatant with a low ammonium (NH₄⁺) concentration. The present invention also provides methods forcultivating cells in cell culture conditions to express a high molecularweight vWF with a high specific activity.

In one aspect, the present invention provides a cell culture solutionfor producing high molecular weight, recombinant vWF protein, the cellculture solution comprising: a cell culture medium comprising a copperconcentration of at least about 2.4 μg/L; a cell culture supernatantcomprising an ammonium concentration of less than 10 mM; and a pluralityof cells expressing highly multimeric vWF protein, wherein the vWFprotein comprises a specific Ristocetin activity of at least about 30mU/μg.

In one specific embodiment of the cell cultures described above, thecell culture solution comprises a medium supplement comprising copper.

In one specific embodiment of the cell cultures described above, themedium supplement comprises a hydrolysate, optionally a soy hydrolysate.

In one specific embodiment of the cell cultures described above, themedium supplement comprises a copper salt, a copper chelate, or acombination thereof.

In one specific embodiment of the cell cultures described above, thecopper salt is selected from the group consisting of copper sulfate,copper acetate, copper carbonate, copper chloride, copper hydroxide,copper nitrate, and copper oxide.

In one specific embodiment of the cell cultures described above, thecopper concentration is at least about 4 μg/L.

In one specific embodiment of the cell cultures described above, thecopper concentration is from about 2.4 μg/L to about 20 μg/L.

In one specific embodiment of the cell cultures described above, the vWFprotein comprises about 14 to about 22 dimers.

In one aspect, the present invention provides a method of producing ahigh molecular weight, recombinant vWF protein, the method comprisingthe steps of: a) providing a culture of cells comprising a nucleic acidencoding recombinant vWF protein; b) expressing vWF protein in the cellsunder cell culture conditions comprising a cell culture mediumcomprising a copper concentration of at least about 2.4 μg/L and a cellculture supernatant comprising an ammonium concentration less than about10 mM, wherein the vWF protein is highly multimeric vWF protein andcomprises a specific Ristocetin activity of at least about 30 mU/μg.

In one specific embodiment of the methods described above, the cells aremammalian cells.

In one specific embodiment of the methods described above, the cells arefrom a continuous cell line.

In one specific embodiment of the methods described above, the cells areCHO cells.

In one specific embodiment of the methods described above, the copperconcentration is at least about 4 μg/L.

In one specific embodiment of the methods described above, the copperconcentration is from about 2.4 μg/L to about 20 μg/L.

In one specific embodiment of the methods described above, therecombinant vWF protein has a specific Ristocetin Cofactor activity ofat least about 50 mU/μg.

In one specific embodiment of the methods described above, therecombinant vWF protein has a specific Ristocetin Cofactor activity fromabout 30 mU/μg to about 100 mU/μg.

In one specific embodiment of the methods described above, therecombinant vWF protein comprises about 14 to about 22 dimers.

In one aspect, the present invention provides a high molecular weight,recombinant vWF protein produced by a process, the process comprisingthe steps of: a) providing a culture of cells comprising a nucleic acidencoding recombinant vWF protein; and b) expressing vWF protein in thecells under cell culture conditions comprising a cell culture mediumcomprising a copper concentration of at least 2.4 μg/L and a cellculture supernatant comprising an ammonium concentration less than 10mM, wherein the vWF protein is highly multimeric vWF protein andcomprises a specific Ristocetin activity of at least about 30 mU/μg.

In one specific embodiment of the rVWF compositions described above, therecombinant vWF protein has a specific Ristocetin Cofactor activity ofat least about 50 mU/μg.

In one specific embodiment of the rVWF compositions described above, therecombinant vWF protein has a specific Ristocetin Cofactor activity fromabout 30 mU/μg to about 100 mU/μg.

In one specific embodiment of the rVWF compositions described above, therecombinant vWF protein comprises about 14 to about 22 dimers.

In one aspect, the present invention provides a cell culture solutionfor producing high molecular weight, recombinant vWF protein, the cellculture solution comprising: a cell culture medium comprising a copperconcentration of at least about 2.4 μg/L; a cell culture supernatantcomprising an ammonium concentration of less than 10 mM; and a pluralityof cells expressing highly multimeric vWF protein, wherein the vWFprotein comprises about 14 to about 22 dimers and a specific Ristocetinactivity of at least about 30 mU/μg.

In one aspect, the present invention relates to cell culture conditionsfor producing recombinant high molecular weight vWF that is in highlymultimeric form with a high specific activity. The cell cultureconditions of the present invention can include, for example, a cellculture medium with an increased copper concentration and cell culturesupernatant with a low ammonium (NH₄ ⁺) concentration. The presentinvention also provides methods for cultivating cells in cell cultureconditions to express a high molecular weight vWF with a high specificactivity.

In one aspect, the present invention includes a cell culture solutionfor producing high molecular weight, recombinant vWF, comprising a cellculture medium comprising a copper concentration of at least about 2.4μg/L; a cell culture supernatant comprising an ammonium concentration ofless than 10 mM; and a plurality of cells expressing highly multimericvWF comprising about 14 to about 22 dimers and a specific Ristocetinactivity of at least about 30 mU/μg. In one embodiment of the cellculture solutions described above, at least 10% of the rVWF is presentin a high molecular weight VWF multimer of more than 10 dimers. In aspecific embodiment, at least 15% of the rVWF is present in a highmolecular weight VWF multimer of more than 10 dimers. In anotherspecific embodiment, at least 20% of the rVWF is present in a highmolecular weight VWF multimer of more than 10 dimers. In anotherspecific embodiment, at least 25% of the rVWF is present in a highmolecular weight VWF multimer of more than 10 dimers. In anotherspecific embodiment, at least 30% of the rVWF is present in a highmolecular weight VWF multimer of more than 10 dimers. In certainembodiments, the copper concentration can be at least about 4 μg/L orthe copper concentration can range from about 2.4 μg/L to about 20 μg/L.In some embodiments, the cell culture media comprise a medium supplementcomprising copper. In certain embodiments, the medium supplement cancomprise a hydrolysate or a copper salt, copper chelate, or acombination thereof. In some embodiments, the copper salt can includecopper sulfate, copper acetate, copper carbonate, copper chloride,copper hydroxide, copper nitrate, or copper oxide. In certainembodiments, the cells that can be from a continuous cell line and caninclude mammalian cells, such as CHO cells. In some embodiments, therecombinant vWF has a specific Ristocetin Cofactor activity of at leastabout 50 mU/μg, or the specific Ristocetin Cofactor activity can rangefrom about 30 mU/μg to about 100 mU/μg.

In another aspect, the present invention includes a method of producinga high molecular weight, recombinant vWF, comprising a) providing aculture of cells; b) introducing a nucleic acid sequence coding for vWF;c) selecting the cells carrying the nucleic acid sequence; and, d)expressing vWF in the cells under cell culture conditions comprising acell culture medium comprising a copper concentration of at least about2.4 μg/L and a cell culture supernatant comprising an ammoniumconcentration less than about 10 mM, wherein the vWF is highlymultimeric vWF comprising about 14 to about 22 dimers and a specificRistocetin activity of at least about 30 mU/μg. In one embodiment of thecell culture supernatant described above, at least 10% of the rVWF ispresent in a high molecular weight VWF multimer of more than 10 dimers.In a specific embodiment, at least 15% of the rVWF is present in a highmolecular weight VWF multimer of more than 10 dimers. In anotherspecific embodiment, at least 20% of the rVWF is present in a highmolecular weight VWF multimer of more than 10 dimers. In anotherspecific embodiment, at least 25% of the rVWF is present in a highmolecular weight VWF multimer of more than 10 dimers. In anotherspecific embodiment, at least 30% of the rVWF is present in a highmolecular weight VWF multimer of more than 10 dimers. In someembodiments, the cells that can be from a continuous cell line and caninclude mammalian cells, such as CHO cells. In certain embodiments, thecopper concentration can be at least about 4 μg/L or the copperconcentration can range from about 2.4 μg/L to about 20 μg/L. In someembodiments, the cell culture media comprise a medium supplementcomprising copper. In certain embodiments, the medium supplement cancomprise a hydrolysate or a copper salt, copper chelate, or acombination thereof. In some embodiments, the copper salt can includecopper sulfate, copper acetate, copper carbonate, copper chloride,copper hydroxide, copper nitrate, or copper oxide. In some embodiments,the recombinant vWF has a specific Ristocetin Cofactor activity of atleast about 50 mU/μg, or the specific Ristocetin Cofactor activity canrange from about 30 mU/μg to about 100 mU/μg.

In yet another aspect, the present invention includes a high molecularweight, recombinant vWF produced by a process, comprising the steps of:a) providing a culture of cells; b) introducing a nucleic acid sequencecoding for vWF; c) selecting the cells carrying the nucleic acidsequence; and, d) expressing vWF in the cells under cell cultureconditions comprising a cell culture medium comprising a copperconcentration of at least 2.4 μg/L and a cell culture supernatantcomprising an ammonium concentration less than 10 mM, wherein the vWF ishighly multimeric vWF comprising about 14 to about 22 dimers and aspecific Ristocetin activity of at least about 30 mU/μg. In oneembodiment of the cell culture supernatant described above, at least 10%of the rVWF is present in a high molecular weight VWF multimer of morethan 10 dimers. In a specific embodiment, at least 15% of the rVWF ispresent in a high molecular weight VWF multimer of more than 10 dimers.In another specific embodiment, at least 20% of the rVWF is present in ahigh molecular weight VWF multimer of more than 10 dimers. In anotherspecific embodiment, at least 25% of the rVWF is present in a highmolecular weight VWF multimer of more than 10 dimers. In anotherspecific embodiment, at least 30% of the rVWF is present in a highmolecular weight VWF multimer of more than 10 dimers. In someembodiments, the cells that can be from a continuous cell line and caninclude mammalian cells, such as CHO cells. In certain embodiments, thecopper concentration can be at least about 4 μg/L or the copperconcentration can range from about 2.4 μg/L to about 20 μg/L. In someembodiments, the cell culture media comprise a medium supplementcomprising copper. In certain embodiments, the medium supplement cancomprise a hydrolysate or a copper salt, copper chelate, or acombination thereof. In some embodiments, the copper salt can includecopper sulfate, copper acetate, copper carbonate, copper chloride,copper hydroxide, copper nitrate, or copper oxide. In some embodiments,the recombinant vWF has a specific Ristocetin Cofactor activity of atleast about 50 mU/μg, or the specific Ristocetin Cofactor activity canrange from about 30 mU/μg to about 100 mU/μg.

In one aspect, the present invention provides a composition comprisingrecombinant Von Willebrand Factor (rVWF) having a specific ristocetincofactor activity of at least 30 mU/μg. In a preferred embodiment, thecomposition has a specific ristocetin cofactor activity of at least 40mU/μg. In a more preferred embodiment, the composition has a specificristocetin cofactor activity of at least 50 mU/μg. In a more preferredembodiment, the composition has a specific ristocetin cofactor activityof at least 60 mU/μg. In a more preferred embodiment, the compositionhas a specific ristocetin cofactor activity of at least 70 mU/μg. In yeta more preferred embodiment, the composition has a specific ristocetincofactor activity of at least 80 mU/μg.

In one embodiment of the compositions described above, at least 10% ofthe rVWF in the composition is present in a high molecular weight VWFmultimer of more than 10 dimers. In a specific embodiment, at least 15%of the rVWF is present in a high molecular weight VWF multimer of morethan 10 dimers. In another specific embodiment, at least 20% of the rVWFis present in a high molecular weight VWF multimer of more than 10dimers. In another specific embodiment, at least 25% of the rVWF ispresent in a high molecular weight VWF multimer of more than 10 dimers.In another specific embodiment, at least 30% of the rVWF is present in ahigh molecular weight VWF multimer of more than 10 dimers.

In one embodiment of the compositions described above, the compositioncomprises a culture supernatant. In one specific embodiment the culturesupernatant is a mammalian cell culture supernatant. In a more specificembodiment, the mammalian cell culture supernatant is a CHO cellsupernatant.

In one embodiment of the compositions described above, the rVWF isexpressed in a cell culture comprising at least 2.4 μg/L copper. In aspecific embodiment, the cell culture comprises at least 4 μg/L copper.In a more specific embodiment, the culture comprises between 2.4 μg/Land 20 μg/L copper. In one embodiment, the copper is provided as acopper salt, a copper chelate, or a combination thereof. In a specificembodiment, the copper salt is selected from the group consisting ofcopper sulfate, copper acetate, copper carbonate, copper chloride,copper hydroxide, copper nitrate, and copper oxide.

In one embodiment of the compositions described above, the cell cultureis a batch culture.

In one embodiment of the compositions described above, the cell cultureis a continuous culture. In a specific embodiment, the continuousculture is performed in chemostatic mode. In another specificembodiment, the continuous culture is performed in perfusion mode.

In one embodiment of the compositions described above, the level of NH4+in the culture is maintained at a concentration below 4 mM.

In one embodiment of the compositions described above, the cell densityof the culture is maintained at less than 2.5×10⁶ cells per mL.

In one embodiment of the compositions described above, the cell densityof the culture is maintained at less than 2.0×10⁶ cells per mL.

In one embodiment of the compositions described above, the culture ismaintained at less than 1.5×10⁶ cells per mL.

In one embodiment of the compositions described above, the rVWF isco-expressed with recombinant Factor VIII (rFVIII). In a specificembodiment, a majority of the co-expressed rFVIII has been removed. In amore specific embodiment, the ratio of rVWF to rFVIII in the compositionis at least 10:1.

In one embodiment of the compositions described above, the compositionis formulated for pharmaceutical administration. In a specificembodiment, the composition is formulated for intravenous, subcutaneous,or intramuscular administration.

In one embodiment of the compositions described above, the compositionis lyophilized.

In yet another aspect, the present invention includes a high molecularweight, recombinant vWF produced by a process, comprising the steps of:a) providing a culture of cells; b) introducing a nucleic acid sequencecoding for vWF; c) selecting the cells carrying the nucleic acidsequence; and, d) expressing vWF in the cells under cell cultureconditions comprising a cell culture medium comprising a copperconcentration of at least 2.4 μg/L and a cell culture supernatantcomprising an ammonium concentration less than 10 mM, wherein the vWF ishighly multimeric vWF comprising about 14 to about 22 dimers and aspecific Ristocetin activity of at least about 30 mU/μg. It isunderstood that all of the embodiments and concentrations described inthe “Cell Culture Media” and “Methods of Producing Recombinant vWF”sections above can apply here.

The recombinant vWF of the present invention can include a highmolecular weight recombinant vWF protein having a high specificactivity. In one embodiment, the vWF of the present invention is ahighly multimeric form of vWF. In some embodiments, the highlymultimeric form of vWF includes at least up to about 14 dimers and inother embodiments at least up to about 22 dimers. In yet otherembodiments, the highly multimeric form of vWF can range from about 10to about 20 dimers, or from about 15 to about 25 dimers, or from about20 to about 40 dimers. In certain embodiments, the recombinant vWF iscomparable to plasmatic vWF.

As described herein, the present invention provides the surprisingresult that increased copper concentration in cell culture media canproduce high molecular weight vWF with high specific activity. Cellculture media comprising a copper concentration, e.g., greater thanabout 2.4 μg/L can increase the yield of recombinant multimeric vWF, ascompared to media without copper. In certain embodiments, the percentageof multimeric vWF (i.e., rVWF comprising at least 2 dimers) can begreater than about 50%, or greater than about 75%, or greater than about90%. The multimeric distribution of the vWF can be analyzed usingstandard techniques such as, e.g., in Agarose electrophoresis undernon-reducing conditions.

As provided herein, the recombinant vWF produced by the methods of thepresent invention can have a high specific activity, e.g., a highspecific Ristocetin Cofactor activity. In one embodiment, therecombinant vWF produced by the methods of the present invention caninclude a specific Ristocetin Cofactor activity of at least 30 mU/μg andin another embodiment at least 50 mU/μg. In other embodiments, thespecific Ristocetin Cofactor activity can range from about 30 mU/μg toabout 100 mU/μg or from about 50 mU/μg to about 100 mU/μg.

B. Recombinant ADAMTS13 (rA13)

The ADAMTS proteins (i.e., ADAMTS-1 to ADAMTS-20) are a family ofsecreted zinc metalloproteinases that share a common modular domainorganization (for review, see, Flannery C. R., Front Biosci. 2006 Jan.1; 11:544-69). All of the ADAMTS protein share a common core domainarchitecture, consisting of a signal peptide, followed by a prodomain, azinc-dependent metalloproteinase catalytic domain, a disintegrin-likedomain, a thrombospondin type I repeat, a cysteine-rich domain, and aspacer domain (Apte S.S., J Biol Chem. 2009 Nov. 13; 284(46):31493-7).Additionally, all but ADAMTS-4 contain at least one more thrombospondintype I repeat domain, and many of the ADAMTS protein contain one or moreadditional ancillary domains. Notably, it has been reported that allADAMTS protein appear to contain at least one calcium binding site andat least one zinc binding site located within the metalloproteinasecatalytic domain (Andreini et al., J. Proteome Res., 2005,4 (3), pp881-888).

Biological roles for ADAMTS proteins have been reported for variousdiseases and conditions, including, Antiangiogenesis, Renal interstitialfibrosis, Bone remodeling, Ovarian folliculogenesis, Atherosclerosis,Urogenital development, and Tumor growth/remodeling (ADAMTS-1);Ehler-Danlos syndrome type 7C and Bovine dermatopraxis (ADAMTS-2);Arthritis, Atherosclerosis, and Tendinopathy (ADAMTS-4); Arthritis andGlioblastoma (ADAMTS-5); Arthritis (ADAMTS-7); Antiangiogenesis, Brainmalignancy, Arthritis, and Atherosclerosis (ADAMTS-8); Arthritis(ADAMTS-9, -12); Thrombotic thrombocytopenic purpura (ADAMTS-13); andAntithrombosis/stroke (ADAMTS18) (for review, see, Lin and Liu, OpenAccess Rheumatology Research and Reviews 2009:1 121-131).

Recombinant ADAMTS13 (A13) has been expressed before in mammalian cells,however the specific activity varies widely dependent on the cellculture conditions. It has been found that many commercially availableculture mediums are not sufficient for expression of rA13 with highspecific activities, expressed as the ratio of activity, measured byFRETS-VWF73 assay, to antigen content, as determined by ELISA. In oneaspect, the methods provided herein are based on several advantageousfindings that allow for cell-culture expression of rA13 having increasedlevels of total and specific activity.

Accordingly, due to the shared structure-function relationship betweenthe ADAMTS family of secreted metalloproteinases, the methods providedby the present invention allow for the expression of all ADAMTS proteinsin cell culture and recovery from the cell medium.

In one aspect, the present invention provides a composition comprisingrecombinant ADAMTS13 (rA13) having a specific FRETS-VWF activity of atleast 1600 mU/μg.

In one embodiment of the compositions described above, the rA13 has aspecific FRETS-VWF activity of at least 800 mU/μg.

In one embodiment of the compositions described above, the compositioncomprises a culture supernatant. In a specific embodiment, the culturesupernatant is a mammalian cell culture supernatant. In a more specificembodiment, the mammalian cell culture supernatant is a CHO cellsupernatant.

In one embodiment of the compositions described above, the rA13 isexpressed in a cell culture comprising at least 1 μg/L copper. In aspecific embodiment, the cell culture comprises at least 2 μg/L copper.In a more specific embodiment, the culture comprises between 2 μg/L and20 μg/L copper.

In one embodiment of the compositions described above, the copper isprovided as a copper salt, a copper chelate, or a combination thereof.In a specific embodiment, the copper salt is selected from the groupconsisting of copper sulfate, copper acetate, copper carbonate, copperchloride, copper hydroxide, copper nitrate, and copper oxide.

In one embodiment of the compositions described above, the cell cultureis a batch culture.

In one embodiment of the compositions described above, the cell cultureis a continuous culture. In a specific embodiment, the continuousculture is performed in chemostatic mode. In another specificembodiment, the continuous culture is performed in perfusion mode.

In one embodiment of the compositions described above, the level of NH₄⁺ in the culture is maintained at a concentration below 5 mM.

In one embodiment of the compositions described above, the level of NH₄⁺ in the culture is maintained at a concentration below 4 mM.

In one embodiment of the compositions described above, the cell densityof the culture is maintained at less than 4.0×10⁶ cells per mL. In aspecific embodiment, the cell density of the culture is maintained atless than 3.0×10⁶ cells per mL. In a specific embodiment, the celldensity of the culture is maintained at less than 2.0×10⁶ cells per mL.In a more specific embodiment, the cell density of the culture ismaintained at less than 1.5×10⁶ cells per mL.

In one embodiment of the compositions described above, the compositionis formulated for pharmaceutical administration. In a specificembodiment, the composition is formulated for intravenous, subcutaneous,or intramuscular administration.

In one embodiment of the compositions described above, the compositionis lyophilyzed.

VI. FORMULATIONS

In one aspect, the formulations comprising the recombinant therapeuticproteins rVWF or rA13 of the present invention are lyophilized prior toadministration. Lyophilization is carried out using techniques common inthe art and should be optimized for the composition being developed[Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res.13:243-9 (1996)].

Methods of preparing pharmaceutical formulations can include one or moreof the following steps: adding a stabilizing agent as described hereinto said mixture prior to lyophilizing, adding at least one agentselected from a bulking agent, an osmolarity regulating agent, and asurfactant, each of which as described herein, to said mixture prior tolyophilization. A lyophilized formulation is, in one aspect, at leastcomprised of one or more of a buffer, a bulking agent, and a stabilizer.In this aspect, the utility of a surfactant is evaluated and selected incases where aggregation during the lyophilization step or duringreconstitution becomes an issue. An appropriate buffering agent isincluded to maintain the formulation within stable zones of pH duringlyophilization.

The standard reconstitution practice for lyophilized material is to addback a volume of pure water or sterile water for injection (WFI)(typically equivalent to the volume removed during lyophilization),although dilute solutions of antibacterial agents are sometimes used inthe production of pharmaceuticals for parenteral administration [Chen,Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)].Accordingly, methods are provided for preparation of reconstitutedrecombinant VWF compositions comprising the step of adding a diluent toa lyophilized recombinant VWF composition of the invention.

The lyophilized material may be reconstituted as an aqueous solution. Avariety of aqueous carriers, e.g., sterile water for injection, waterwith preservatives for multi dose use, or water with appropriate amountsof surfactants (for example, an aqueous suspension that contains theactive compound in admixture with excipients suitable for themanufacture of aqueous suspensions). In various aspects, such excipientsare suspending agents, for example and without limitation, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents are a naturally-occurring phosphatide, forexample and without limitation, lecithin, or condensation products of analkylene oxide with fatty acids, for example and without limitation,polyoxyethylene stearate, or condensation products of ethylene oxidewith long chain aliphatic alcohols, for example and without limitation,heptadecaethyl-eneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example and without limitation, polyethylene sorbitanmonooleate. In various aspects, the aqueous suspensions also contain oneor more preservatives, for example and without limitation, ethyl, orn-propyl, p-hydroxybenzoate.

To administer compositions to human or test animals, in one aspect, thecompositions comprises one or more pharmaceutically acceptable carriers.The phrases “pharmaceutically” or “pharmacologically” acceptable referto molecular entities and compositions that are stable, inhibit proteindegradation such as aggregation and cleavage products, and in additiondo not produce allergic, or other adverse reactions when administeredusing routes well-known in the art, as described below.“Pharmaceutically acceptable carriers” include any and all clinicallyuseful solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like,including those agents disclosed above.

The pharmaceutical formulations are administered intravenously, orally,topically, transdermally, parenterally, by inhalation spray, vaginally,rectally, or by intracranial injection. The term parenteral as usedherein includes subcutaneous injections, intravenous, intramuscular,intracisternal injection, or infusion techniques. Administration byintravenous, intradermal, intramusclar, intramammary, intraperitoneal,intrathecal, retrobulbar, intrapulmonary injection and or surgicalimplantation at a particular site is contemplated as well. Generally,compositions are essentially free of pyrogens, as well as otherimpurities that could be harmful to the recipient.

Single or multiple administrations of the compositions are carried outwith the dose levels and pattern being selected by the treatingphysician. For the prevention or treatment of disease, the appropriatedosage depends on the type of disease to be treated, the severity andcourse of the disease, whether drug is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the drug, and the discretion of the attending physician.

In one aspect, formulations of the invention are administered by aninitial bolus followed by a continuous infusion to maintain therapeuticcirculating levels of drug product. As another example, the inventivecompound is administered as a one-time dose. Those of ordinary skill inthe art will readily optimize effective dosages and administrationregimens as determined by good medical practice and the clinicalcondition of the individual patient. The frequency of dosing depends onthe pharmacokinetic parameters of the agents and the route ofadministration. The optimal pharmaceutical formulation is determined byone skilled in the art depending upon the route of administration anddesired dosage. See for example, Remington's Pharmaceutical Sciences,18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712,the disclosure of which is hereby incorporated by reference. Suchformulations influence the physical state, stability, rate of in vivorelease, and rate of in vivo clearance of the administered agents.Depending on the route of administration, a suitable dose is calculatedaccording to body weight, body surface area or organ size. Appropriatedosages may be ascertained through use of established assays fordetermining blood level dosages in conjunction with appropriatedose-response data. The final dosage regimen is determined by theattending physician, considering various factors which modify the actionof drugs, e.g. the drug's specific activity, the severity of the damageand the responsiveness of the patient, the age, condition, body weight,sex and diet of the patient, the severity of any infection, time ofadministration and other clinical factors. By way of example, a typicaldose of a recombinant vWF of the present invention is approximately 50U/kg, equal to 500 μg/kg. As studies are conducted, further informationwill emerge regarding the appropriate dosage levels and duration oftreatment for various diseases and conditions.

VII. METHODS OF TREATMENT

The present invention further contemplates methods of treating a patientin need of rVWF or rA13 produced according to the methods describedherein. Such methods of treatment can include administration ofpharmaceutical formulations comprising the recombinant rA13 or the highmolecular weight, recombinant vWF of the present invention.

In another aspect, the present invention provides methods fortherapeutic or prophylactic treatments comprising the administration ofan rVWF or rA13 composition provided herein. Generally, for therapeuticapplications, formulations are administered to a subject with a diseaseor condition associated with ADAMTS13 or VWF dysfunction or otherwise inneed thereof, in a “therapeutically effective dose.” Formulations andamounts effective for these uses will depend upon the severity of thedisease or condition and the general state of the patient's health.Single or multiple administrations of the formulations may beadministered depending on the dosage and frequency as required andtolerated by the patient.

In one embodiment, the present invention provides methods of treating orpreventing a disease or condition associated with an ADAMTS13 or VWFdysfunction. In a further embodiment, the pharmaceutical formulationscomprising recombinant vWF can be administered to treat diseases relatedto vWF, such as von Willebrand's disease or hemophilia. In anotherembodiment, the invention provides methods of treating or preventing adisease or condition associated with the formation and/or presence ofone or more thrombus, comprising the administration of a rA13composition provided herein. In another embodiment, the inventionprovides methods of disintegrating one or more thrombus in a subject inneed thereof. In yet other embodiments, the invention provides methodsof treating or preventing an infarction in subject in need thereof.Generally, the methods provided by the invention comprise administeringan rADAMTS13 composition as provided herein to a subject in needthereof.

Non-limiting examples of disorders associated with the formation and/orthe presence of one or more thrombus are hereditary thromboticthrombocytopenic purpura (TTP), acquired TTP, arterial thrombosis, acutemyocardial infarction (AMI), stroke, sepsis, and disseminatedintravascular coagulation (DIC).

Non-limiting examples of disorders associated with an infarction,include without limitation, myocardial infarction (heart attack),pulmonary embolism, cerebrovascular events such as stroke, peripheralartery occlusive disease (such as gangrene), antiphospholipid syndrome,sepsis, giant-cell arteritis (GCA), hernia, and volvulus.

VIII. EXAMPLES

The present invention will now be further illustrated in the followingexamples, without being limited thereto.

Example 1

Continuous cell culture experiments were performed using cultures of arecombinant CHO cell line expressing vWF. The basal medium was DMEM/F12,which contained about 0.3 μg/L of Cu²⁺. The medium was supplemented withsoy hydrolysate and copper sulfate (CuSO₄.5H₂O) to bring the finalcopper concentration in the medium to greater than at least 2.4 μg/L.

Recombinant CHO cells expressing vWF were cultivated by continuous cellcultures so that the ammonium levels (NH₄ ⁺) were kept at aconcentration less than about 10 mM. It was found that productionsystems providing a continuous supply of medium (e.g., perfusion orchemostat cultures) were preferable because conventional batch or fedbatch techniques produced high NH₄ ⁺ concentrations at the end of theculture. At the end of the culture, the highly multimeric vWF wasisolated and the specific Ristocetin Cofactor activity of the vWF wasmeasured.

Example 2

Recombinant Factor VIII (rFVIII) and Von Willebrand Factor (rVWF) wereco-expressed in batch cultures of GD8/6 cells to determine the effect ofthe composition of the culture medium on VWF expression and activity.Briefly, GD8/6 cells were batch cultivated in BAV-SP medium composed ofa modified DMEM/F12 basal powder (Table 5) and additional supplementsalso containing 4 g/L of soy hydrolysate, see Table 6, with and withoutadditional copper supplementation. To test the effect of low copperconcentraions on rVWF expression and activity, basal BAV-SP media wasused. The basal media contained 0.3 μg/L copper and was supplementedwith soy hydrolysate, which contributed an additional 0.7 μg/L copper,as determined experimentally, providing a final copper concentration of1.0 μg/L. As a comparision, the BAV-SP media used for the batch cultureswas further supplemented with an additional 3.3 μg/L of Cu', providing afinal copper concentration of 4.3 μg/L, to determine the effect highcopper concentrations on VWF expression and activity.

TABLE 5 Composition of BAV-SP Culture Media BAV-SP-Medium Componentsmg/L Amino Acids L-Alanine 4.45 L-Arginine HCl 147.50 L-Asparagine-H₂O30.21 L-Aspartic Acid 6.65 L-Cystèine HCl—H₂O 32.55 L-Cystine 2HCl 57.35L-Glutamic Acid 7.35 Glycine 18.75 L-Histidine-H₂O HCl 31.48Hydroxy-L-Proline L-Isoleucine 54.47 L-Leucine 59.05 L-Lysine HCl 91.25L-Methionine 17.24 L-Phenylalanine 35.48 L-Proline 52.24 L-Serine 26.25L-Threonine 53.45 L-Tryptophan 29.01 L-Tyrosine 2Na 2H2O 55.79 L-Valine52.85 Vitamins Ascorbic Acid 3.499 Biotin 0.0035 Choline Chloride 8.980D-Ca-Pantothenate 2.240 Folic Acid 2.650 I-Inositol 12.600 Nicotinamide2.020 Pyridoxine HCl 2.031 Riboflavin 0.219 Thiamine HCl 2.170 VitaminB12 0.680 Anorganic Salts Calcium Chloride (CaCl2) 116.600 CopperSulfate (CuSO₄—5H₂O) 0.0013 Ferric Nitrate (Fe(NO₃)3—9H₂O) 0.050 FerrousSulfate (FeSO₄—7H₂O) 0.417 Magnesium Chloride (MgCl2) 28.640 MagnesiumSulfate (MgSO4) 48.840 Potassium Chloride (KCl) 311.800 Sodium Chloride(NaCl) 6995.500 Sodium phosphate (Na2HPO4) 71.020 Sodium phosphate(NaH2PO4) 62.500 Zinc Sulfate Heptahydrate (ZnSO4—7H2O) 0.432 Sodiumselenite 0.0131 Others D-Glucose 3151 Linoleic Acid 0.042 Lipoic Acid0.105 Putrescine 2HCl 0.081 Thymidine 0.365 Hypoxantine Na 2.390 SodiumPyruvate 55.000

TABLE 6 Composition of BAV-SP Supplement Componets for final formulationL-Glutamine 600.00 Ethanolamine 1.530 Synperonic F68 250.000 SodiumBicarbonate (NaHCO3) 2000.000 Soy peptone 4000.00 Total formulation18596.8

GD8/6 cells expressing rVWF were grown in either low copper media (Table7) or high copper media (Table 8) for 7 days. After 2 days the cultureswere subcultured to perform a batch culture for a period of 5 days.Samples of the culture supernatant were tested daily for rVWF content(vWF ELISA), total (Ristocetin) and specific (Specific Activity) via aristocetin cofactor assay. Various culture parameters, including cellcount, cell viability, and ammonium concentration were also monitoreddaily (except for batch days 3 and 4).

Unexpectedly, cell cultures grown under high copper concentrationsproduced supernatants containing significantly higher total and specificrVWF activity (compare results in Table 7 and Table 8). For example, atbatch day 4, the cell culture grown under high copper concentrationcontained 1.52 IU rVWF activity/mL, as compared to 0.2 IU rVWFactivity/mL for the low copper culture. This is despite the fact thatthe low copper cell culture produced nearly twice the amount of rVWF asthe high copper cell culture. Furthermore, the specific activity of thesupernatant obtained from the high copper culture was more than 13 timesgreater than that of the low copper culture supernatant (831 mU/10 μgrVWF v. 62 mU/10 μg rVWF).

As seen in Table 7 and Table 8, the total and specific ristocetincofactor activity of the high copper culture was twice that of the lowcopper culture at batch day 1. Furthermore, in contrast to thesubsequent increases in activity observed in the high copper culture, noincrease in the amount of ristocetin cofactor activity was seen afterbatch day 1 for the low copper cell culture. Consistent with thisresult, agarose gel electrophoresis analysis of the rVWF multimer staterevealed that a low concentration of high molecular weight rVWF,relative to the concentration of low molecular weight rVWF species, waspresent in the supernatant of the low copper cell culture at batch day1, and that the relative concentration further decreased over time(FIGS. 1A and 1B). In contrast, supplementation of the culture mediumwith Cu²⁺ resulted in a consistent formation of ristocetin cofactor(RiCoF) active antigen through the fourth day. Consistently, no loss inthe amount of stable high molecular weight rVWF multimers occurredthrough day 4 of the batch culture (FIGS. 1A and 1B). The densitometricresults of the agarose electrophoresis gel shown in FIG. 1B revealedthat under low copper conditions the culture was only able to produce arVWF population in which more than 10% (i.e., 16.3%) of the rVWF waspresent in molecules having more than 10 dimers for one day of the batchculture (i.e. “day 3” sample in Lane 2 of the agarose gel of FIG. 1A),and notably, this population fell to only 4% at day 5 of the batchculture (“day 7” sample in Lane 6). In contrast under high copperconditions the relative amount of vWF multimers with more than 10 dimersis consistently around 30% through day 4 of the batch culture (“day 3”to “day 6” in Lanes 7-10; 28% to 31.4%).

Notably, beginning at batch day 5 of the high copper batch culture, whenNH₄ ⁺ levels exceeded 100 mg/L (greater than about 5.0 mM), theexpression of additional antigen (18.3 to 35.4 μg/L; compare batch days4 and 5 of Table 8) did not result in a concomitant increase in theRiCoF activity present in the supernatant. Consistent with this result,the level of high molecular weight rVWF multimers at batch day 5 (day 7of the culture) decreased relative to the concentration of low molecularweight rVWF multimers. Also only on batch day 5 (“day 7” sample of theculture in Lane 11) the relative amount of the vWF with more than 10dimers is reduced to 21.4%.

Taken together, the data provided above demonstrate that thesupplementation of copper concentrations in cell cultures expressingrVWF dramatically increases the total and specific rVWF ristocetincofactor activity, as well the stable production of high molecularweight rVWF multimers. Furthermore, the data show a correlation betweenthe presence of high NH₄ ⁺ concentrations in the cell culture and theloss of rVWF ristocetin cofactor activity and high molecular weight rVWFmultimer production.

TABLE 7 Expression of rVWF in mammalian cell culture performed in batchmode using BAV-SP media with a low copper concentration (1.0 μg/L). vWFSpecific batch Cell Count NH₄ ⁺ Viability ELISA Ristocetin Actifity day[10E6 cells/ml] [mg/l] [%] [μg/ml] [IU/mL] mU/10 μg 0 0.42  21 98.8 n.d.n.d. n.d. 1 0.78  43 n.d.  6.3 0.23 365 2 1.24  64 99.1 12.8 0.23 180 31.86 n.d. n.d. 22.7 0.21 93 4 2.49 n.d. n.d. 32.2 0.20 62 5 3.11 10698.3 44.4 0.20 45

TABLE 8 Expression of rVWF in mammalian cell culture performed in batchmode using BAV-SP media with a high copper concentration (4.3 μg/L). vWFSpecific batch Cell Count NH₄ ⁺ Viability ELISA Ristocetin Actifity day[10E6 cells/ml] [mg/l] [%] [μg/ml] [IU/mL] mU/10 μg 0 0.40  21 99.3 n.d.n.d. n.d. 1 0.71  42 n.d. 4.8 0.40 833 2 1.23  63 99.5 8.7 0.62 713 31.85 n.d. n.d. 15.0 1.07 713 4 2.54 n.d. n.d. 18.3 1.52 831 5 3.32 10998.9 35.4 1.55 438

Example 3

Recombinant Factor VIII (rFVIII) and Von Willebrand Factor (rVWF) wereco-expressed in continuous cultures of GD8/6 cells operated underchemostatic conditions to determine the effect of the composition of theculture medium on VWF expression and activity. Briefly, GD8/6 cells werecultivated in BAV-SP medium containing 4 g/L of soy hydrolysate with andwithout copper supplementation as described in Example 2. To test theeffect of low copper concentraions on rVWF expression and activity,basal BAV-SP media was used. The basal media contained 0.3 μg/L copperand was supplemented with soy hydrolysate, which contributed anadditional 0.7 μg/L copper, providing a final copper concentration of1.0 μg/L. As a comparision, the BAV-SP media used for the batch cultureswas further supplemented with an additional 3.3 μg/L of Cu²⁺, providinga final copper concentration of 4.3 μg/L, to determine the effect highcopper concentrations on VWF expression and activity. Cultures grown inthe presence of high and low copper concentrations were cultivated underboth high (2.8×10⁶ cells/mL) and low (appr. 1.4×10E06 cells/mL) celldensities.

As before, samples of the culture supernatant were tested for rVWFcontent (vWF ELISA), total (Ristocetin) and specific (Specific Activity)activity via a ristocetin cofactor assay. Various culture parameters,including cell count, cell viability, and ammonium concentration werealso monitored. Data were generated from the steady state phase of weeks2 and 3 of the chemostat cultures (Table 9 to Table 13).

TABLE 9 Mean data for rVWF expression in chemostatic cell culture duringweeks 2 and 3. Cell Count vWF specific Cell Copper [10E6 NH4+ ViabilityELISA Ristocetin actifity Count Concentration cells/mL] [mM] [%] [gg/ml][IU/mL] [mU/10 μg] high low 2.88 3.88 97.74 44.56 0.10 21.62 high high2.79 4.04 98.25 38.38 0.19 53.11 low low 1.55 3.33 98.63 18.96 0.1050.16 low high 1.43 3.17 98.59 11.76 0.70 598.76

TABLE 10 rVWF expression in chemostatic cell culture under high cellcount, low copper conditions during weeks 2 and 3. vWF specific CellCount NH4+ Viability ELISA Ristocetin actifity Day [10E6 cells/mL] [mM][%] [m/ml] [IU/mL] [mU/10 μg]  8 2.54 3.7 98.20 39.8 0.095 23.86934673 9 3.02 4.2 97.40 10 2.97 3.9 97.90 41.3 0.095 23.00242131 11 2.78 132.91 3.8 97.60 41.7 0.095 22.78177458 14 2.90 3.8 97.40 15 3.05 3.997.70 44.7 0.095 21.25279642 16 2.99 3.8 98.30 17 2.76 3.9 97.40 55.30.095 17.17902351 2.88 3.88 97.74 44.56 0.10 21.62

TABLE 11 rVWF expression in chemostatic cell culture under high cellcount, high copperc onditions during weeks 2 and 3. vWF specific CellCount NH4+ Viability ELISA Ristocetin actifity Day [10E6 cells/mL] [mM][%] [μg/ml] [IU/mL] [mU/10 μg]  8 2.52 3.9 98.30 31.8 0.35 110.0628931 9 2.92 4.3 98.50 10 2.80 4.2 98.60 37.4 0.32 85.56149733 11 2.57 132.81 3.9 98.50 37.6 0.095 25.26595745 14 2.92 3.9 97.80 15 2.77 3.997.80 43.0 0.095 22.09302326 16 2.72 4.0 98.30 17 3.04 4.1 98.20 42.10.095 22.56532067 2.79 4.04 98.25 38.38 0.19 53.11

TABLE 12 rVWF expression in chemostatic cell culture under low cellcount, low copper conditions during weeks 2 and 3. vWF specific CellCount NH4+ Viability ELISA Ristocetin actifity Day [10E6 cells/mL] [mM][%] [μg/ml] [IU/mL] [mU/10 μg]  8 1.61 3.3 99.10 19.3 0.095 49.22279793 9 1.65 3.3 98.00 10 1.49 3.3 98.90 18.6 0.095 51.07526882 11 1.58 131.58 3.4 98.10 18.1 0.095 52.48618785 14 1.56 3.3 99.30 15 1.52 3.398.50 18.9 0.095 50.26455026 16 1.50 3.3 98.40 17 1.50 3.4 98.70 19.90.095 47.73869347 1.55 3.33 98.63 18.96 0.10 50.16

TABLE 13 rVWF expression in chemostatic cell culture under low cellcount, high copper conditions during weeks 2 and 3. Cell Count vWFspecific [10E6 NH4+ Viability ELISA Ristocetin actifity Day cells/mL][mM] [%] [μg/ml] [IU/mL] [mU/10 μg]  8 1.46 3.2 98.80 11.6 0.73629.3103448  9 1.45 3.2 98.20 10 1.37 3.1 98.90 11.0 0.7 636.3636364 111.43 13 1.33 3.2 98.10 11.1 0.68 612.6126126 14 1.39 3.2 97.20 15 1.513.2 99.70 12.4 0.69 556.4516129 16 1.49 3.2 98.60 17 1.43 3.2 99.20 12.70.71 559.0551181 1.43 3.17 98.59 11.76 0.70 598.76

As shown in FIG. 2A, supernatant harvested from continuous rVWF cellculture grown at low cell densities and high copper concentrationscontained high specific activity (average of 600 mU/10 μg), whilesupernatants harvested from rVWF cell cultures grown at high celldensities in the presence of high or low copper concentrations and rVWFcell cultures grown at low cell density in the presence of low copperconcentration contained low specific activities (less than 100 mU/10μg). Consistent with the results observed for batch cultures, FIG. 2Bshows that continuous mammalian cell cultures expressing rVWF with highspecific activities have lower NH₄ ⁺ concentrations than do culturesproducing rVWF with low specific activities. This data furtherstrengthen the correlation between NH₄ ⁺ concentration in the cellculture and the specific activity of rVWF produced by the culture.Notably, the combination of high copper concentration and low ammoniumconcentration in the cell culture allowed for the productionsignificantly improved rVWF activity.

Consistent with this result, agarose gel electrophoresis analysis of therVWF multimer state of chemostat cultures (FIG. 6) revealed that onlysupernatants from cultures operated at high copper and low cell densityand thus low ammonium resulted in a consistent expression highmultuimeric vWF (about 23% to 27% on CST day 8, 17 and 24 in Lanes 4, 8,and 12, respectively). All other conditions do not reach consistentlyhigh amounts of more than 10% of vWF with more than 10 dimers over anextended cultivation period.

Example 4

To determine the effect of culture media copper concentration on theexpression and specific activity of rA13, mammalian cell culturesexpression rA13 were grown for 4 weeks under chemostatic continuousculture conditions in ADAMTS13 medium comprising a modified DMEM/F12basal media BESP845 and additional supplements (Table 14) containingcopper concentrations ranging from 0.66 μg/L (without additional coppersupplementation) and with additional copper supplementation to 4 μg/L.As shown in Table 15, increasing copper concentration in the cellculture media resulted in a significant increase in volumetric (P) andspecific (q) productivity expressed as the total rA13 activity producedper liter culture and day and the total rA13 activity produced per celland day, respectively.

TABLE 14 Composition of ADAMTS13 medium. DMEM/F12 BESP845 mg/L AminoAcids L-Alanine 13.3500 L-Arginine HCl 147.5000 L-Asparagine-H₂O 45.1600L-Aspartic Acid 19.9500 L-Cystéine HCl—H₂O 32.5500 L-Cystine 2HCl102.3500 L-Glutamic Acid 22.0500 Glycine 26.2500 L-Histidine-H₂O HCl51.4800 L-Isoleucine 74.4700 L-Leucine 119.0500 L-Lysine HCl 146.2500L-Methionine 100.0000 L-Phenylalanine 60.4800 L-Proline 63.7400 L-Serine36.7500 L-Threonine 53.4500 L-Tryptophan 29.0100 L-Tyrosine 2Na 2H2O75.7900 L-Valine 82.8500 salts Calcium Chloride (CaCl2) 116.6000 CopperSulfate (CuSO₄—5H₂O) 0.0026 Ferric Nitrate (Fe(NO₃)3—9H₂O) 0.0500Ferrous Sulfate (FeSO₄—7H₂O) 1.0170 Magnesium Chloride (MgCl2) 28.6400Magnesium Sulfate (MgSO4) 48.8400 Potassium Chloride (KCl) 311.8000Sodium Chloride (NaCl) 5495.5000 Na2HPO4 Anhydrous 213.0200 NaH2PO4Anhydrous 54.3500 Zinc Sulfate Heptahydrate (ZnSO4—27H2O) 0.4320 Sodiumselenite•anhydrous 0.0087 Vitamins Ascorbic Acid 3.4990 Biotin 0.2035Choline Chloride 26.9800 D-Ca-Pantothenate 6.2400 Folic Acid 6.6500I-Inositol 36.6000 Nicotinamide 7.0200 Pyridoxine HCl 6.0310 Riboflavin0.6590 Thiamine HCl 6.5100 Vitamin B12 2.6800 miscellaneous D-Glucose5000.0000 Linoleic Acid 0.0420 Lipoic Acid 1.0050 Putrescine 2HCl 3.6810Thymidine 0.3650 Hypoxantine Na 2.3900 Sodium Pyruvate 55.0000 DMEM/F12BESP845: Total 12738.3 ADAMTS-13 Medium Supplements L-Glutamin 1300.0000Pluronic F68 1000.0000 Etanolamin 1.5300 ZnSo4*7 H2O 1.0000Na-Hydrogencarbonat 1500.0000 Supplements Total 3802.5 Total amount ofIngridients 16540.8

To determine if increased copper concentrations affected the integrityof the expressed rA13, supernatant harvested from rA13 cell culturesgrown in media containing 0.66 μg/L, 1 μg/L, and 4 μg/L copper wasexamined by SDS-PAGE analysis. As seen in FIG. 3A (silver stain) andFIG. 3B (anti-A13 western blot), no obvious change in product quality bygel electrophoresis was observed. Specifically, no increase in the levelof truncated 170 kD or other low MW rA13 variants and no otheradditional or increased HCP bands resulted from rA13 expression in thepresence of increased copper concentrations.

To estimate an optimal concentration of copper on the activity of rA13,an extrapolation of data from Table 15 of P Frets versus copperconcentration (FIG. 4) shows that optimum effect is likely reached atabout 2 μg/L, with a conservative estimation of a negative effect aboveabout 4 μg/L.

TABLE 15 Expression of rA13 in mammalian cell culture performed inchemostatic continuous culture mode using media containing variablecopper concentrations. 10 L Scale ZZ - NC q Frets Spec. R&D Experiment[10E6 P Frets [U/(10E9 Activity Mean of 4 CST weeks cells/mL] [U/(L*d)]cells*d)] U/mg 0.66 μg/L Cu²⁺   1.35 1322 1028 1759 1 μg/L Cu²⁺ 1.641962 1247 1690 4 μg/L Cu²⁺ 2.26 2960 1338 1768

Based on the results obtained above, an additional experiment wasperformed, which compared the expression of rA13 in cell culture mediacontaining 0.66 μg/L copper with 2.0 μg/L copper. As shown in Table 16,supplementation of the basal cell media with copper, to a finalconcentration of 2.0 μg/L copper, resulted in a significant increase involumetric (P) and specific (q) productivity expressed as the total rA13activity produced per liter culture and day and the total rA13 activityproduced per cell and day, respectively, over a period of 8 weeks. Thespecific data for each week of rA13 production in the two cultures isshown in FIG. 5. From these data, it is clear that coppersupplementation has a measurable beneficial effect on cell metabolism,specific growth rate and rA13 productivity.

TABLE 16 Expression of rA13 in mammalian cell culture performed inchemostatic continuous culture mode using media containing variablecopper concentrations. ZZ - NC q Frets Spec. 10 L Scale [10E6 P Frets[U/(10E9 Activity Mean of 8 CST weeks cells/mL] [U/(L*d)] cells*d)] U/mg0.66 μg/L Cu²⁺ 1.29 1049 821 1862   2 μg/L Cu²⁺ 2.17 2470 1146 1749

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1.-45. (canceled)
 46. A composition comprising isolated solublerecombinant Von Willebrand Factor (rVWF), wherein the composition has anNH4+ concentration of below 4 mM or below 10 mM, wherein the compositioncontains at least 0.5 IU ristocetin cofactor activity per mL, andwherein at least 20% of the isolated soluble rVWF in the composition ispresent in a high molecular weight VWF multimer of more than 10 dimers.47. The composition of claim 46, wherein the composition has a rVWFspecific ristocetin cofactor activity of at least 30 mU/μg rVWF, atleast 40 mU/μg rVWF, or at least 50 mU/μg rVWF.
 48. The composition ofclaim 46, wherein the composition has a rVWF specific ristocetincofactor activity of at least 60 mU/μg rVWF, at least 70 mU/μg rVWF, orat least 80 mU/μg rVWF.
 49. The composition of claim 46, wherein atleast 20% of the isolated soluble rVWF is present in a high molecularweight VWF multimer of 14 to 22 dimers.
 50. The composition of claim 46,wherein the composition has an NH4+ concentration of below 10 mM, below9 mM, or below 8 mM.
 51. The composition of claim 46, wherein thecomposition has an NH4+ concentration of below 7 mM, below 6 mM, orbelow 5 mM.
 52. The composition of claim 46, wherein the composition hasan NH4+ concentration of below 4 mM, 3 mM, 2 mM, or 1 mM.
 53. Thecomposition of claim 46, wherein the composition further comprisesrecombinant Factor VIII (rFVIII).
 54. The composition of claim 53,wherein the ratio of rVWF to rFVIII is at least 10:1.
 55. A cell culturesolution comprising a cell culture media and one or more cellscomprising a nucleic acid encoding a recombinant Von Willebrand Factor(rVWF) protein, wherein the cell culture media comprises a copperconcentration of at least 2.4 μg/L and an ammonium concentration ofbelow 4 mM or below 10 mM.
 56. The cell culture solution of claim 55,wherein the copper in the cell culture media is provided as a coppersalt, a copper chelate, or a combination thereof.
 57. The cell culturesolution of claim 56, wherein the copper salt is selected from the groupconsisting of copper sulfate, copper acetate, copper carbonate, copperchloride, copper hydroxide, copper nitrate, and copper oxide.
 58. Thecell culture solution of claim 55, wherein the cell culture media is aprotein free culture media.
 59. The cell culture solution of claim 55,wherein the one or more cells are mammalian cells.
 60. The cell culturesolution of claim 59, wherein the mammalian cells are CHO cells.
 61. Thecell culture solution of claim 55, wherein the cell culture mediacontains from 5 mg/L to 10 mg/L nicotinamide.
 62. The cell culturesolution of claim 55, wherein the cell culture media contains about 7mg/L nicotinamide.
 63. The cell culture solution of claim 55, whereinthe cell culture media comprises an NH4+ concentration of below 10 mM,below 9 mM, below 8 mM, below 7 mM, or below 6 mM.
 64. The cell culturesolution of claim 55, wherein the cell culture media comprises an NH4+concentration of below 5 mM, below 4 mM, below 3 mM, or below 2 mM. 65.The cell culture solution of claim 55, wherein the cell culture mediacomprises an NH4+ concentration of below 1 mM.